Her first effort as a thespian was as her brother’s assistant in magic shows the two would concoct for their family. She later attended the Ted Liss Acting Studio in Chicago and Harand Camp Adult Theater Seminar in Elkhart Lake, Wisconsin. Of her experience with Liss, she said:

Audiences first caught a glimpse of Madsen in a bit part she landed as Lisa in the teen sex comedy Class. She portrayed a role as cellist named Madeline in Electric Dreams (1984) which was the first film release by Virgin Films Production Company. She was cast as Princess Irulan in David Lynch’s science fiction epic Dune (1984).

Madsen first became popular with audiences in 1986 with her portrayal of a Catholic schoolgirl who fell in love with a boy from a prison camp in Duncan Gibbons’s Fire with Fire. As beauty queen Dixie Lee Boxx, she was the sexy romantic interest of minor-league baseball manager Cecil “Stud” Cantrell (William Petersen) in the made-for-cable Long Gone (1987). She appeared as Maddie Hayes’ cousin in the final season of Moonlighting.

She also starred in the 1992 slasher film Candyman. During the filming of Candyman Madsen was hypnotised so that her pupils would remain dilated while she acted out certain scenes. The director Bernard Rose wanted the character to not have a typical screaming style of when confronted by the villain. Madsen stated in an interview for the DVD in 2004 that there were some days during filming that she did not remember much due to the hypnotism. She told her director that she did not want to be hypnotised after the initial few experiences.

A couple of provocative film appearances followed: 1990′s The Hot Spot with Don Johnson, directed by Dennis Hopper, and the equally steamy Third Degree Burn with Treat Williams. She was also co-host of the TV series Unsolved Mysteries in 1999, the show’s final season on CBS.

Madsen turned in a solid performance in a small but key role in the Francis Ford Coppola drama The Rainmaker (1997) starring Matt Damon. Film critic Roger Ebert said that Madsen had a “strong scene,” while reviewer James Berardinelli noted that “the supporting cast is solid, with turns from… Virginia Madsen as a witness for the plaintiff”. Madsen had spent more than twenty years in minor films before her breakout critically acclaimed performance in Sideways (2004). The role catapulted her onto the fabled Hollywood A-list. Her first major role after Sideways was opposite Harrison Ford in Firewall. She later appeared in Robert Altman’s A Prairie Home Companion, in a key role as the angel. She co-starred with Jim Carrey in The Number 23 and Billy Bob Thornton in The Astronaut Farmer; both films opened in North America on February 23, 2007.

She voiced Queen Hippolyte, mother of Wonder Woman, in the 2009 animated Wonder Woman film.

Madsen has made numerous television appearances including: Star Trek: Voyager, CSI: Miami, Dawson’s Creek, The Practice, Frasier, and other series. She starred opposite Ray Liotta in CBS’s short-lived crime drama series Smith. She also made many appearances final season on Monk.