In October 2005, Stoner, along with Lucio Cecchinello's team, reportedly had an agreement to move to the MotoGP class in the upcoming season with support from Yamaha. After the season ended, he received an offer from the Honda Pons team and tested the Honda RC211V bike with them at Valencia. However, in December 2005, Stoner re-signed with Cecchinello's team after Honda Pons failed to secure sponsorship for the upcoming season. The team then made an agreement with LCR Honda to run the RC211V for Stoner in 2006.

As a rookie satellite rider, Stoner took the pole position in just his second MotoGP race, but crashed several times during the season. He finished in 8th position in the championship, with his best result being a second place at the Turkish Grand Prix. He was leading the race until he was overtaken on the final corner by Marco Melandri.

Stoner secured a ride with the Factory Ducati Team for the 2007 season, joining Loris Capirossi on the new 800cc Ducati Desmosedici GP7. Stoner started off his Ducati career on a high note with a first premier-class win in the Qatar opener, after a tense battle with Valentino Rossi. Stoner took ten race wins and six pole positions (including winning three of the first four), took him to his first GP title, by a margin of 125 points (equivalent to five victories) over Dani Pedrosa, which he built during the second half of the season. His worst finish was a 6th place at Motegi, which was all he needed to clinch the title that day, taking the first premier class title for an Italian or a non-Japanese manufacturer since Phil Read's title for MV Agusta in 1974. Stoner was named Young Australian of the Year for his 2007 performance.

In 2008, Stoner took the #1 decat on his bike. Stoner opened the 2008 season with a victory at Qatar, before a run of two races without a podium. He returned to the podium with a second place at Mugello, before starting a run of seven successive pole positions. He turned three of them into successive victories-a lights-to-flag win at Donington, leading every lap at Assen six days later, and recovering from a huge Friday crash at Sachsenring to win in the wet after Dani Pedrosa crashed, moving to within 20 points of the championship lead. However, successive crashes while fighting for the lead at Mazda Raceway Laguna Seca (where he remounted to finish second to Valentino Rossi), Brno and Misano ensured that he could not defend the title successfully. Stoner finished the 2008 season with six wins and was runner-up to Rossi with 280 points, the highest number of points ever gained without taking the title at the time.

Stoner remained with Ducati for the 2009 season with new teammate Nicky Hayden, with a further option for a 4th season in 2010. A strong start to the season left Stoner in a three-way battle with the Fiat Yamaha duo of Rossi and Jorge Lorenzo, before he was struck by a mystery illness which caused him to feel tired long before the end of races, leaving him 16 points behind Rossi and 7 behind Lorenzo after the US Grand Prix at Laguna Seca on 5 July.

Stoner was initially diagnosed with anaemia and an inflammation of the stomach lining. Stoner later disputed the diagnosis, however, and, after continuing to struggle with the condition, he announced on 10 August 2009 that he would miss rounds 11, 12 and 13 in Brno, Indianapolis and Misano, respectively, in an attempt to recover from the illness, he was subsequently diagnosed as lactose intolerant. Mika Kallio was chosen as Stoner's replacement for the three races. Stoner returned to racing late in the 2009 season, placing second in the Portuguese Grand Prix and an emphatic first in the Australian Grand Prix, which he led throughout. At interview following the Australian Grand Prix, Stoner said that he experienced none of the premature tiredness that had dogged him earlier in the 2009 season. He followed this up with another first place in the wet at the Malaysian Grand Prix. At the last round of 2009 at Valencia, Stoner dominated all practice and qualifying sessions to take pole, only to crash on cold tyres on the warm-up lap and miss the race. Stoner ended the season with four victories, and eight podiums in total, leading to a fourth-place finish in the riders' championship.

At the test held immediately following the Valencia round, Stoner was once again fastest while testing the new 2010 version of the Desmosedici. However, Rossi was fastest in five of the six pre-season tests. Stoner qualified on pole for the season opener in Qatar, and was leading the race when he crashed out, later acknowledging that this was his own mistake. He also crashed out of round 3 at Le Mans, this time attributing the crash to the front of the bike unloading when not running at maximum pace. His first podium of the year came at Assen, despite struggling with arm pump late in the race.

It was not until the thirteenth race of the season, the inaugural Aragon Grand Prix, that Stoner achieved his first victory. His victory in Aragon started a run of three victories in four races, as he also won the delayed Japanese Grand Prix, and won for the fourth consecutive year at Phillip Island. He eventually finished fourth in the riders' championship once again. With Rossi having fallen out of favour with Yamaha following Lorenzo's championship-winning season and Honda no longer willing to play second-fiddle to another Japanese manufacturer, an intense game of musical-chairs ensued in the MotoGP paddock that saw several of the top riders switch teams, Stoner among them. For 2011, Stoner joined Honda Racing Corporation after four years at Ducati Corse, where he was replaced by Valentino Rossi.

Stoner raced with the Repsol Honda Racing Team in 2011, with teammates Dani Pedrosa and Andrea Dovizioso. In preseason testing in Malaysia, Stoner was quickest in all three sessions, closely followed by Pedrosa and reigning world champion Jorge Lorenzo. Stoner won the season-opening race in Qatar from pole position, and had been quickest in each of the free practice sessions held before qualifying. Stoner took pole position for the Spanish Grand Prix, but had been running second behind Marco Simoncelli when he was taken out by the Ducati of Valentino Rossi in wet conditions. Stoner won three out of the first five rounds of the season, with victories in Le Mans and Catalunya to add to his Qatar victory. Stoner added victories at Silverstone in damp conditions, and Laguna Seca, to hold a 20-point lead over Jorge Lorenzo with eight races to go in the season.

Stoner proposed boycotting the Japanese Grand Prix out of fears for his health from radiation from the Fukushima Daiichi Nuclear Power Plant] even though all the independent scientific experts including the World Health Organization and Australian Radiation Protection and Nuclear Safety Agency stated that it is safe to live permanently 80 km (50 mi) or more from the plant.

Stoner won the World Championship for the second time at his home round at Phillip Island, Australia. On his 26th birthday, Stoner won his ninth race of the season from his eleventh pole, and with his only challenger Jorge Lorenzo ruled out of the race due to a hand injury suffered in warm-up, Stoner finished the weekend with an unassailable 65-point lead. His victory in the Australian MotoGP was his fifth in succession in his home race dating back to 2007 which made him the only rider to have won at Phillip Island during the 800cc era of MotoGP. He also was the only rider other than Marc Marquez or Lorenzo in the premier class to have won the championship in the 2010s.

Stoner started the season with wins at Jerez, and Estoril, both tracks he had not won a MotoGP race at before; his victory in Estoril allowed him to take the championship lead. By finishing fourth at the Catalan Grand Prix, Stoner finished off the podium for the first time in fourteen months. He won the Dutch TT at Assen to move back level on points with Lorenzo, who was taken-out by Álvaro Bautista on the first lap. This put Stoner even in points with Lorenzo before a final-lap retirement at the Sachsenring, while battling teammate Dani Pedrosa. Stoner finished only eighth at the Italian Grand Prix after running off-circuit, later describing that he was "not comfortable" on the bike, but followed that up with a fourth win of the season at Laguna Seca.

At the next race, Stoner crashed heavily during the qualifying session for the Indianapolis Grand Prix, suffering torn ligaments in his ankle but was declared fit to race the following day. He finished fourth in the race, 2.5 seconds behind third-placed Andrea Dovizioso. Stoner then elected to have surgery on his ankle, ruling him out of action for three races which essentially put him out of contention for the championship. Stoner's priority then was to be fully recovered for his home race in Australia, and he was slated to return at the Japanese Grand Prix in October. Upon returning, he finished fifth in Japan and third in Malaysia, before winning his home race for the sixth successive season at Phillip Island. The win gave him an undefeated record on Bridgestone tyres at the circuit.