Mamola grew up in Northern California and showed early talent as a musician before turning to motorcycle racing at age 12, idolising fellow Californian Kenny Roberts. He began in dirt track racing and earned Yamaha sponsorship at 14. In 1978, he won the AMA 250cc road racing title, drawing comparisons to Roberts and earning the nickname "Baby Kenny." His strong showing at the 1979 Transatlantic Trophy match races, where he finished as the second-highest points scorer behind fellow American Mike Baldwin ahead of former world champion Barry Sheene, announced his international potential.
Mamola entered the 1979 Grand Prix season in the 250cc class but switched to the premier 500cc class mid-season following an injury to teammate Mike Baldwin. Despite competing in only half the races, he took two second-place finishes, ending the year fourth in the 250cc class and eighth in the 500cc standings.
Mamola earned a full Suzuki factory contract for 1980. He won the Belgian Grand Prix that year, becoming — at 20 years and 239 days — the youngest Grand Prix premier class winner in history at that time, surpassing a record set by Mike Hailwood in 1961. Freddie Spencer subsequently broke the record in 1982. Mamola also won the British Grand Prix before mechanical failure at the final race at the Nürburgring cost him the championship. He finished the season second behind Roberts.
In 1981, Mamola led the championship with two wins and two second places before Gallina-Suzuki rider Marco Lucchinelli claimed four victories in five races to take the title. His crew chief Jeremy Burgess — later Valentino Rossi's chief for seven world championships — believed Mamola would have won had he not been restricted to Dunlop tyres against Lucchinelli's preferred Michelins. For the second consecutive year, Mamola finished runner-up.
After a difficult 1982 and a competitive 1983 — third behind Freddie Spencer and Kenny Roberts — Mamola lost his Suzuki ride when the factory withdrew from Grand Prix racing. Honda signed him as a replacement for the injured Spencer in 1984. Riding the NS500 and then the new NSR500, Mamola won three of the last five races of the season but again finished the year in second place, behind Eddie Lawson. It was his third championship runner-up result.
In 1985 and 1986, Mamola rode for the Kenny Roberts Yamaha team, winning at Belgium in 1986 and recording six podiums, though he sparked controversy at the French Grand Prix by performing a stoppie just before the finish line while lying second. In 1987, he won three Grands Prix and scored nine podiums but finished behind Wayne Gardner for his fourth and final runner-up season.
After Roberts replaced Mamola and Mike Baldwin with Wayne Rainey and Kevin Magee for the 1988 season, Mamola joined Cagiva. He spent three years helping develop the Cagiva C589, hampered throughout by limited funding. Following the 1990 season he sat out 1991 entirely, returning in 1992 for a final season on a privately supported Yamaha. He took his last podium at the Hungarian Grand Prix that year — a third place — and retired at the end of the season ranked tenth in the world championship.
At the 1985 San Marino Grand Prix, Mamola produced one of the most remarkable rider saves in the sport's history. After his rear tyre lost and then instantly regained traction, he was thrown forward over the handlebars. Gripping the bars with both hands while both legs flailed off the right side of the machine, he held the motorcycle upright until he could swing his left leg back into position and regain control, continuing the race.
After retiring from competition, Mamola worked as a test rider for Yamaha before becoming a television commentator covering MotoGP for international broadcasters. In 1986, while still racing, he had become involved with Save the Children, an experience that led him to co-found Riders for Health — an organisation that supplies and maintains motorcycles, ambulances and four-wheel vehicles to deliver healthcare in seven African countries. The organisation also trains local riders in vehicle maintenance. Mamola later established Two Wheels for Life as a successor initiative. He has raised funds at Grand Prix events by auctioning rider-donated memorabilia and offering passenger experiences on a two-seater Ducati MotoGP machine.