Agostini was born in Brescia, the son of a wealthy, Italian industrialist. Agostini's father originally didn't approve of his son's motorcycle racing career. He did everything he could to persuade his son not to race. Agostini would have to steal away to compete, first in hill climb events and then in road racing.
Eventually his father came to terms with his racing and he won the 1963 Italian 175cc championship aboard a Morini. He got his break when Morini factory rider, Tarquinio Provini left the team to ride for Benelli. Count Alfonso Morini hired the young Agostini to ride for him. In 1964, Agostini would win the Italian 350cc title and proved his ability by finishing fourth in the Italian Grand Prix at Monza.
These results caught the eye of Count Domenico Agusta who signed Agostini to ride for his MV Agusta squad as Mike Hailwood's team-mate. Agostini then fought a season-long battle with Honda's Jim Redman for the 1965 350cc world championship. He seemed to have the title won when he led the final round in Japan at Suzuka when, his bike failed him handing the title to Redman.
Hailwood left to join Honda as he had tired of working for the difficult Count Agusta. Agostini responded by winning the 500cc title seven years in succession for the Italian factory. He would also win the 350cc title six times in succession and won 10 Isle of Man TTs. In 1967 he battled Hailwood in one of the most dramatic seasons in Grand Prix history. Each rider had 5 victories before the championship was decided in Agostini's favor at the last race of the season.
Agostini dropped a bombshell on the Grand Prix world when, after the death of his close friend, Gilberto Parlotti at the 1972 Isle of Man TT, he announced he would never again race at the event because he considered it unsafe. At the time, the TT was the most prestigious race on the motorcycling calendar. Other top riders joined his boycott of the event and by 1976, the event was stricken from the Grand Prix schedule.
Agostini surprised the racing world when he announced that he would ride for Yamaha in 1974. On his first outing for the Japanese factory, he won the prestigious Daytona 200. He went on to claim the 1974 350cc World Championship but injuries and mechanical problems kept him from winning the 500cc crown. He rebounded and won the 1975 500cc title, the first time that a two-stroke machine had claimed the premier class. It would also be his last world title. In 1976, he would win his last Grand Prix, taking an MV Agusta to victory at the Nürburgring. He retired from motorcycle competition after finishing 6th in the 1977 season.
Like John Surtees and Mike Hailwood before him, Agostini began an auto racing career in 1978 but never managed to reach Formula One. He competed in the European Formula 2 series in a Chevron-B42 BMW and British Aurora Formula 1 with his own team and a Williams FW06-1. He ended his auto racing career in 1980.
Ago returned to the motorcycle racing as a Marlboro team manager. As team manager he managed many successful riders such as Kenny Roberts, Freddie Spencer and Eddie Lawson.