Bledel made her TV debut on The WB series Gilmore Girls in 2000. She portrayed Rory Gilmore, the daughter of single mother Lorelai Gilmore. Initially, Rory was a high school student living with her mother, but later moved on to college at Yale University, where she, among other things, worked as the editor of the Yale Daily News. Bledel has said that she feels she can relate in some ways to Rory, both when it comes to some personal traits and her relationship to her own parents. She also sees the family structure and mother-daughter relationship on the show as something to which many people can relate. In 2002, Bledel was voted one of Teen People's "25 Hottest Stars Under 25". She appeared as the main character in the video for Less Than Jake's "She's Gonna Break Soon", lead single from their 2003 album Anthem. The single charted at number 39 on the UK charts. In 2005, she starred in The Sisterhood of the Traveling Pants based on the book by Ann Brashares. She played Lena Kaligaris, a talented artist on a journey with her three best friends, linked over the summer by a pair of magical jeans.

In 2005, Bledel made a shift from previous TV and film characters while portraying Becky, a prostitute, in the movie Sin City. "She's a very professional prostitute. She carries a gun and she kicks ass," said Bledel of Becky. Sin City is shot almost entirely in black-and-white, with splashes of color here and there, such as a red dress, blood or an entirely yellow evil character. In Becky's case, it was her eyes that kept their color, as director Robert Rodriguez claimed that he found Bledel's eyes to be "so amazing I had to keep them blue in the picture." In the summer of 2007, Bledel wrapped filming of The Sisterhood of the Traveling Pants sequel. It has been announced that Bledel will star in The Post Grad Survival Guide as Ryden Malby. She has recently been cast in a new movie entitled The Letters, which is anticipated to be released in 2009. The Letters is a drama / thriller film by director Todd Fjelsted and is currently in pre-production. Bledel is set to star opposite Scott Porter in the upcoming film The Good Guy, also due for a 2009 release. In this, Porter plays a Wall Street businessman whose life begins to fall apart when he helps out a new salesman. Bledel will play Beth, Porter's girlfriend.

• The Post Grad Survival Guide (2009) (post-production) .... Ryden Malby
• The Good Guy (2009) (post-production) .... Beth
  
• The Sisterhood of the Traveling Pants 2 (2008) .... Lena Kaligaris
• "Gilmore Girls" .... Rory Gilmore (154 episodes, 2000-2007)
  ... aka Gilmore Girls: Beginnings (USA: rerun title)
       - Bon Voyage (2007) TV episode .... Rory Gilmore
       - Unto the Breach (2007) TV episode .... Rory Gilmore
       - Lorelai? Lorelai? (2007) TV episode .... Rory Gilmore
       - It's Just Like Riding a Bike (2007) TV episode .... Rory Gilmore
       - Hay Bale Maze (2007) TV episode .... Rory Gilmore
        (149 more)
• Life Is Short (2006) .... Charlotte
• I'm Reed Fish (2006) .... Kate Peterson
• The Sisterhood of the Traveling Pants (2005) .... Lena
  ... aka The Sisterhood of the Travelling Pants (Australia)
• Sin City (2005) .... Becky
  ... aka Frank Miller's Sin City (USA: complete title)
• The Orphan King (2005) .... Dylan
• Bride & Prejudice (2004) .... Georgina 'Georgie' Darcy
  ... aka Balle Balle! Amritsar to L.A. (India: English title)
  ... aka Bride and Prejudice: The Bollywood Musical
  (International: English title: promotional title)
• DysEnchanted (2004) .... Goldilocks
• Tuck Everlasting (2002) .... Winifred 'Winnie' Foster