Slate was born in Milton, Massachusetts to parents Nancy Slate and Ron Slate, a Massachusetts-based poet and author. She has two sisters, Abigail and Stacey, and is Jewish. After graduating from Milton Academy as the valedictorian, she attended Columbia University, where she helped form the improv group Fruit Paunch, and starred in the Varsity Show. Slate graduated from Columbia University in 2004.

Alongside comedian Gabe Liebman, Slate is half of the comedy duo Gabe & Jenny. Their show with Max Silvestri, Big Terrific, was named best new variety show of 2008 by Time Out New York. She met Liebman in 2000 while attending Columbia University. They describe their relationship as a "nonsexual romance", and Slate says "I like to think of us as kind of like Elaine Benes and George Costanza, but we like each other."

Prior to joining SNL, Slate was best known as a regular commentator on many VH1 talking head commentary programs. In early 2009 Slate had made several appearances on the Late Night with Jimmy Fallon recurring sketch 7th Floor West where she played an NBC page, later promoted to Fallon's assistant (also named Jenny), and she also had recurring roles on HBO's Bored to Death (as Stella) and on the FOX sitcom Brothers. Slate also starred in the 2009 short film Obvious Child.

Throughout 2008 and 2009 Slate regularly performed her one-woman show, Jenny Slate: Dead Millionaire, at the Upright Citizens Brigade Theater (UCBT) in New York City. She also performed the stage show at UCBT's Los Angeles division for a number of weeks in early 2010.

In August 2010, Slate co-wrote and voiced the stop-motion short film "Marcel the Shell With Shoes On", directed by Dean Fleischer-Camp, about a shy, shoe-wearing shell, which garnered unexpected viral success, and in turn, may lead to more shorts.

Slate joined the cast of Saturday Night Live for its 35th season in 2009. In her first episode, she accidentally said "fuck" during her debut sketch, which was heard on the live broadcast, however the expletive was removed from all future reruns. On SNL, she impersonated celebrities such as Hoda Kotb, Olympia Snowe, Lady Gaga, Ashley Olsen and Kristen Stewart. Her best known recurring character was Tina-Tina Chaneuse, an infomercial pitchwoman of unspecified Latino heritage who advertises personalized doorbells, car horns and alarm clocks.

In September 2010, it was reported that Slate's contract with SNL would not be renewed for a second season.

Slate lives in Brooklyn, New York and is dating filmmaker Dean Fleischer-Camp.