Guthrie was born in Iowa City, Iowa, and grew up in Miami, Florida after her father, a pilot, took a job with Eastern Air Lines. She earned her own pilot's license at 17. After studying physics at the University of Michigan, where she graduated in 1960, she worked as an aerospace engineer with Republic Aviation. Racing entered her life in 1963 when she began competing on the SCCA circuit in a Jaguar XK140. By 1972 she was racing on a full-time basis, and her sportscar career included two class victories in the 12 Hours of Sebring.
Guthrie entered NASCAR in 1976, finishing fifteenth in the World 600 to become the first woman to compete in a NASCAR Winston Cup superspeedway race. She competed in four additional races that season. In 1977 she made her Daytona 500 debut, finishing twelfth after her engine lost two cylinders with ten laps remaining; she was still awarded Top Rookie honors for the race. Over four seasons in NASCAR's top series she competed in 33 races total, with her best finish of sixth place at Bristol Motor Speedway in 1977 — a result that stood as the best finish by a woman in a top-tier NASCAR race in the modern era, later tied by Danica Patrick in 2014.
Guthrie first attempted to qualify for the Indianapolis 500 in 1976 but did not make the field. Her top practice lap in a back-up car loaned by three-time race winner A.J. Foyt — who was angered by fellow drivers dismissing her on the basis of gender — would have been sufficient to qualify, but she was unable to secure the funding for her own entry. She qualified for and competed in the 1977 Indianapolis 500 in a car entered by Rolla Vollstedt, finishing 29th with engine trouble. In 1978, driving with a fractured wrist sustained in a charity tennis event two days before the race — an injury she concealed from race officials — she finished ninth, her best result in three Indianapolis starts. She competed in 11 Indy car events in total, with a best career finish of fifth.
Guthrie's career unfolded against sustained resistance from an overwhelmingly male racing establishment, and her inability to secure consistent corporate sponsorship cut her time at the top level short. In a 1987 interview she identified the structural problem clearly: sponsors pursuing publicity were choosing male drivers over women even though, as she noted, a successful woman driver would attract many times the attention. She later said her perspective on addressing sexism in motorsport changed when she qualified for the 1977 Indianapolis 500 and took part in the downtown parade, seeing parents holding up their young daughters as a symbol of what had become possible.
In 2011 she signed a petition calling on Saudi King Abdullah to sponsor a Saudi Women's Grand Prix. Her story was the subject of a 2019 ESPN 30 for 30 documentary titled Qualified.
Guthrie's racing helmet and fire suit are held in the collection of the Smithsonian Institution. Her 2005 autobiography, Janet Guthrie: A Life at Full Throttle, received praise from publications including Sports Illustrated. A feature film about her life starring Hillary Swank was announced in 2021.
She has been inducted into the International Women's Sports Hall of Fame (1980), the International Motorsports Hall of Fame (2006), the Indianapolis Motor Speedway Hall of Fame (2006), the Automotive Hall of Fame (2019), the Motorsports Hall of Fame of America (2021), and the NASCAR Hall of Fame (2024). She received the NASCAR Landmark Award in January 2024.