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Guillem was the top-ranking female dancer with the Paris Opera Ballet from 1984 to 1989, before becoming a principal guest artist with the Royal Ballet in London. She is currently performing contemporary dance as an Associate Artist of London's Sadler's Wells Theatre. Her most notable performances have included those in Giselle and in Rudolf Nureyev's stagings of Swan Lake and Don Quixote.
In 1977 at age 11, she begin training at the Paris Opera Ballet School where Claude Bessy, then director of the school, immediately noticed her exceptional capacities and potential, and in 1981 at age 16, she joined the company's corps de ballet. Initially she hated dancing, preferring gymnastics, but after taking part in her show she found that she loved performing. 
In 1983 Guillem won the gold medal at the Varna International Ballet Competition, which later in the year earned her her first solo role, dancing the Queen of the Driads in Rudolf Nureyev's staging of Don Quixote. In December 1984, after her performance in Nureyev's Swan Lake, she became the Paris Opera Ballet's youngest-ever étoile, the company's top-ranking female dancer. In 1987 she performed the lead role in William Forsythe's contemporary ballet In the Middle, Somewhat Elevated with one of her favourite partners, Laurent Hilaire.
In 1988 she was given the title role in a production of Giselle staged by the Royal Ballet to celebrate Nureyev's 50th birthday. Her performance was a success, and in the following year she left Paris for London, to become a freelance performer and one of the Royal Ballet's principal guest artists. Her desire to work independently from a company gained her the nickname Mademoiselle Non. In 1995 Guillem created the dance television program, Evidentia, which won several international awards. In 1998 she staged her own version of Giselle for the Finnish National Ballet, and in 2001 restaged the ballet for La Scala Ballet in Milan.
In 2001 she became the first winner of the Nijinsky Prize for the world's best ballerina, although in her acceptance speech she criticised the "supermarket culture" of such awards. In the same year, she controversially appeared nude and without make-up in a photo-shoot for French Vogue. In 2003 she directed the central section of a Nureyev tribute program, but was criticised for having the dancers perform in front of a giant projected backdrop of Nureyev, which the audience found distracting. By 2006 she had moved from ballet to contemporary dance, working with such performers as Akram Khan as an Associate Artist of the Sadler's Wells Theatre in London, England.
Guillem's repertoire includes Giselle (Giselle), Swan Lake (Odette/Odile), Don Quixote (Kitri), In the Middle, Somewhat Elevated, Romeo and Juliet (Juliet), The Sleeping Beauty (Aurora), Boléro, Cinderella, Notre-Dame de Paris, Raymonda, La Bayadère (Nikiya and Gamzatti), Fall River Legend, Prince of the Pagodas (Princess Rose), Hermann Schmermann, Le Martyre de Saint-Sébastien, and Sacred Monsters (with Akram Khan).
Guillem has received numerous decorations during her career.