In the late 1950s, Lisi did theater at Piccolo Teatro di Milano in I giacobini by Federico Zardi under the direction of Giorgio Strehler. During the 1960s, Lisi did comedies and participated in television dramas that were widely viewed in Italy. Lisi also promoted a toothpaste brand on television with a slogan that would become a catchphrase among Italians: "con quella bocca può dire ciò che vuole" (with such a mouth, she can say whatever she wants).
Ancona-born Lisi began her film career in her teens. Discovered in Rome by two Neapolitan producers, Antonio Ferrigno and Ettore Pesce, she debuted in La corda d'acciaio (The Steel Rope, 1953). Initially, she did musical films, like E Napoli canta (Naples Sings, 1953) and the successful Questa è la vita (Of Life and Love, 1954, with the popular Totò). Nonetheless, her beauty was more valued than her talent, as seen in Le diciottenni (Eighteen Year Olds) and Lo scapolo (The Bachelor) films of 1955. Yet she filled demanding roles, particularly in La donna del giorno (1956), Eva (1962), and the spectacle Romolo e Remo (1961).
To overcome her typecasting playing seductresses, Lisi sought new types of roles, of evil women or of a lover in relationships of disparate age for Example. In those years, she participated in Italian productions, in Casanova 70 and Le bambole (1965), Arabella (1967), and Le dolci signore (1968). Lisi also starred in The Birds, the Bees and the Italians (1965) which shared the Palme d'Or at Cannes that year.
In the early 1970s, she took a temporary hiatus from acting to spend more time with her husband Franco Pesci and their son, Corrado. Nonetheless, Lisi's career underwent a renaissance with a number of projects, including Al di là del bene e del male (1977), Ernesto (1979), and La cicala (1980). For the film La Reine Margot (1994), Lisi's portrayal of Catherine de' Medici won her both the César and Cannes Film Festival awards, along with a Silver Ribbon for Best supporting Actress. In 2002, Lisi starred in Il più bel giorno della mia vita.
The Argentinian band Sumo (led by Luca Prodan) made a song for her, "TV Caliente a.k.a. Virna Lisi" (1986), composed by Luca Prodan, whose brother, the actor Andrea Prodan, appeared with her in the movie I ragazzi di via Panisperna (1988). A Brazilian rock band named Virna Lisi (1989–1997) was named after her. Meilland International SA named a rose after her in 1989.
On 18 December 2014, Lisi died of lung cancer in Rome at age 78.
