She is best known for voicing Lois Griffin in the animated comedy series Family Guy (1999–present), for which she won a Primetime Emmy Award.

She gained further acclaim for starring as Susie Myerson in the comedy-drama series The Marvelous Mrs. Maisel (2017–2023), which earned her two Primetime Emmy Awards.

She had supporting roles in numerous films, including Showtime (2002), Bad Santa (2003), Kicking & Screaming (2005), Little Man (2006), Killers (2010), Ted (2012), A Million Ways to Die in the West (2014), Love the Coopers (2015), and Extinct (2021).

Her maternal grandmother, a Holocaust survivor, was born and raised in Budapest, Hungary, and immigrated to the United States after the Hungarian Revolution of 1956.

In 1997, Borstein became a cast member on the third season of the sketch comedy show MADtv after being scouted by talent agents.

Borstein was best known on MADtv for her character Ms. Swan (owner of the Gorgeous Pretty Beauty Nail Salon); her other recurring characters included Eracist Anne, "Stick Chick" Echo, singer Jasmine Wayne-Wayne, child prodigy Karen Goddard, lounge singer Shaunda, News at Six outside-the-studio reporter Sue Napersville, and Cordo the GAP troll.

While working on MADtv, Borstein met Seth MacFarlane, who was then preparing to launch the animated sitcom Family Guy on Fox.

As well as her role as the voice performer for Lois and several other characters (including a brief appearance as Ms. Swan in a 2005 episode), she is also a producer and staff writer.

Borstein made recurring appearances on Gilmore Girls throughout the show's run, first as the harpist Drella and later as the stylist Miss Celine.

In 2007, it released the DVD Drop Dead Gorgeous (in a Down-to-Earth Bombshell Sort of Way), a recording of a live performance at the Alex Theatre with Teddy Towne as the opening act.

[10] Borstein has made several supporting appearances in such comedic films as Killers (2010), Dinner for Schmucks (2010), Ted (2012), ParaNorman (2012), A Million Ways to Die in the West (2014), and The Angry Birds Movie (2016).

In 2010, Borstein joined the first season staff of the Showtime comedy-drama series Shameless, as a writer and supervising producer.