Mladin began his professional racing career in 1992, immediately winning the Australian Superbike Championship. He made his debut in the 500cc World Championship the following year, riding for the Cagiva factory, though he finished a disappointed thirteenth and was unhappy with how the team managed him.
In 1995, a plane crash threatened to cost him his foot. He recovered and returned to finish second in the Australian Superbike championship that year, demonstrating the resilience that would define his subsequent career.
Mladin joined the AMA Superbike series in 1996 with Yoshimura Suzuki, finishing fourth overall in his debut season. He switched to Fast By Ferracci Ducati for 1997 and finished third in the championship before returning to Suzuki for 1998, where he again placed third. By the time his first title arrived in 1999, he had spent three seasons learning the circuits and the series.
Mladin's breakthrough came in 1999 when he claimed his first AMA Superbike title with Yoshimura Suzuki. He followed it immediately with back-to-back championships in 2000 and 2001, cementing his status as the series' dominant force. After finishing second in 2002 — the year Nicky Hayden claimed the title — Mladin responded with three more consecutive championships in 2003, 2004, and 2005.
Beyond the season standings, Mladin was a three-time winner of the Daytona 200, taking the prestigious race in 2000, 2001, and 2004. He also made wildcard appearances in the Superbike World Championship, taking pole position at Mazda Raceway Laguna Seca in 2003.
Mladin's dominance was finally challenged by his young Yoshimura Suzuki teammate Ben Spies. In 2006, despite winning the final five races of the season after adapting his riding style to compete with Spies, Mladin finished second in the championship. In 2007 he again came second, losing to Spies by a single point in one of the closest title battles in AMA history.
The 2008 season brought controversy. Following a round at Virginia International Raceway, AMA Pro Racing disqualified Mladin after finding his motorcycle's crankshaft had been modified in violation of the rules requiring an essentially stock crankshaft. His team's appeal was dismissed as without merit, and with one round remaining, Spies' points advantage became unassailable. Mladin lost the championship for a third consecutive year.
On 31 July 2009, Mladin announced he would retire at season's end. The announcement came the same weekend he refused to participate in a round at Heartland Park, Kansas, citing concerns about rider safety — a cause he had championed throughout his career. He won the 2009 AMA Superbike Championship before retiring.
In 2024, Mladin was inducted into the AMA Motorcycle Hall of Fame at a ceremony held in Pickerington, Ohio.
Mat Mladin's record of seven AMA Superbike Championships places him alone at the top of the all-time list for American Superbike titles. His 83 career wins and 50 pole positions in the series remain unsurpassed. Although he never competed full-time in MotoGP or the World Superbike Championship, his sustained excellence in American road racing across fourteen seasons established him as one of the most successful motorcycle racers the sport has produced in any domestic series. His relentless advocacy for circuit safety improvements added a dimension to his legacy beyond raw results.