Having run across an ad for an open casting call and attending her first audition, Rodriguez beat 350 other applicants to win her first role in the low-budget 2000 independent film, Girlfight. Rodriguez's performance as Diana Guzman, a troubled teen who decides to channel her aggression by training to become a boxer, was recognized by both critics and audiences, but despite major industry buzz of a possible Academy Award nomination, she did not receive the Oscar nod. Rodriguez did however accumulate several other significant awards and nominations for the role in independent circles, including major acting accolades from the National Board of Review, Deauville Film Festival, Independent Spirit Awards, Gotham Awards, Las Vegas Film Critics Sierra Awards, and many others. The film itself took home top prizes at both the Sundance and Cannes Film Festivals.

Subsequently, she has had notable roles in other successful movies, including The Fast and the Furious, Resident Evil, Blue Crush, and S.W.A.T. In 2004, Rodriguez lent her voice to the video game Halo 2, playing a Marine. She also provided the voice of Liz Ricarro in the Cartoon Network series IGPX. From 2005 to 2006, she played tough cop Ana Lucia Cortez on the television series Lost during the show's second season (the character's first appearance was a flashback on season 1's finale, Exodus: Part 1). In 2006, Rodriguez was featured in her own episode of G4's show Icons.

Her latest film, Battle in Seattle, is currently playing in limited release and she recently wrapped filming alongside the original cast on the fourth installment of the The Fast and the Furious franchise entitled simply Fast & Furious which releases April 3, 2009. Rodriguez can also be seen in James Cameron's upcoming $200 million sci-fi adventure Avatar, due out December 18, 2009. She recently wrapped filming on Trópico de Sangre, an independent film she is co-producing with her production company Cheshire Kat Productions, based on the Dominican Republic's historic Mirabal sisters who were assassinated in 1960 by the Dominican dictator for opposing his rule.

• Avatar (2009) (post-production) .... Trudy Chacon
  ... aka Avatar: An IMAX 3D Experience (USA: IMAX version)
• Trópico de sangre (2009) (post-production) .... Minerva Mirabal
• Fast & Furious (2009) (completed) .... Letty Marciano
• "Lost" .... Ana-Lucia Cortez (25 episodes, 2005-2009)
       - The Lie (2009) TV episode .... Ana-Lucia Cortez
       - Live Together, Die Alone (2006) TV episode (credit only) .... Ana-Lucia Cortez
       - Three Minutes (2006) TV episode (credit only) .... Ana-Lucia Cortez
       - ? (2006) TV episode .... Ana-Lucia Cortez
       - Two for the Road (2006) TV episode .... Ana-Lucia Cortez
        (20 more)
• A Cat's Tale (2008) (V) (voice) .... Jujube
  ... aka Cats: The Movie! (USA: DVD title)
• Gardens of the Night (2008) .... Lucy
• Battle in Seattle (2007) .... Lou
• The Breed (2006) .... Nicki
• BloodRayne (2005) .... Katarin
• "IGPX: Immortal Grand Prix" .... Liz Ricarro (1 episode, 2005)
       - Time to Shine (2005) TV episode (voice: English version) .... Liz Ricarro
• Control (2004) .... Teresa
• Halo 2 (2004) (VG) (voice) .... Marine
• Driv3r (2004) (VG) (voice) .... Calita
• True Crime: Streets of LA (2003) (VG) (voice) .... Rosie Velasco
• S.W.A.T. (2003) .... Chris Sanchez
• Blue Crush (2002) .... Eden
  ... aka Blue Crush (Germany)
• Resident Evil (2002) .... Rain Ocampo
  ... aka Resident Evil (France) (Germany)
• The Fast and the Furious (2001) .... Letty
  ... aka The Fast and the Furious (Germany)