Sprinkle was born Ellen F. Steinberg on July 23, 1954, in Philadelphia, Pennsylvania, to a Russian-Jewish mother and a Polish-Jewish father. She is known as the "prostitute and porn star turned sex educator and Artist." Her best known theater and performance art piece is her Public Cervix Announcement, in which she invites the audience to "celebrate the female body" by viewing her cervix with a speculum and flashlight. She also performed The Legend of the Ancient Sacred Prostitute, in which she did a "sex magic" masturbation ritual on stage. She has toured one-woman shows internationally for 17 years, some of which were titled Post Porn Modernist, Annie Sprinkle's Herstory of Porn, Hardcore from the Heart. She then performed two-women shows with Beth Stephens titled Exposed; Experiments in Love, Sex, Death and Art, Dirty Sex Ecology, Earthly: An Ecosex Bootcamp, and Ecosex Walking Tour.

Sprinkle has also long championed sex worker rights and health care. She has also worked as a prostitute from 1973-1992.

As Ellen Steinberg, Sprinkle began working at the ticket booth at Tucson's Plaza Cinema at 18, when Deep Throat was playing. The film was busted, and when Steinberg had to appear in court as a witness, she met and fell in love with Deep Throat's Director, Gerard Damiano, and became his mistress, following him to New York City, where she lived for twenty-two years. Not long after becoming Damiano's mistress, Steinberg began working in porn herself and, at that time, started calling herself “Annie.” (Below can be found more details about the stage name she adopted.) Annie's first porn movie was Teenage Deviate, which was released in 1975. Perhaps her best known mainstream porn featured role was in Deep Inside Annie Sprinkle (co-directed by Sprinkle and sexploitation veteran Joseph W. Sarno) which was the No. 2 grossing porn film of 1981.

In 1991, Sprinkle created the Sluts and Goddesses workshop, which became the basis for her 1992 production The Sluts and Goddesses Video Workshop – Or How To Be A Sex Goddess in 101 Easy Steps, which was co-produced and co-directed with videographer Maria Beatty, and featured music by Composer Pauline Oliveros. Sprinkle pioneered new genres of sexually explicit film and video such as edu-porn, gonzo, post porn, xxx docudrama, art porn, and feminist erotica. Sprinkle has also presented many sex workshops with fellow sex facilitator Barbara Carrellas, with whom she presented the stage production Metamorphosex.

She starred in Nick Zedd's experimental films War Is Menstrual Envy (1992), Ecstasy in Entropy (1999), and Electra Elf: The Beginning (2005).

Sprinkle's work has always been about sexuality, with a political, spiritual, and artistic bent. In December 2005, she committed to doing seven years of art projects about love with her art collaborator and eventual wife, Beth Stephens. They called this their Love Art Laboratory. Part of their project was to do an experimental art wedding each year, and each year had a different theme and color. The seven-year structure was adapted to their project by invitation of Artist Linda M. Montano. Sprinkle and Stephens have done twenty-one art weddings, eighteen with ecosexual themes. They married the Earth, Sky, Sea, Moon, Appalachian Mountains, the Sun, and other non-human entities in nine different countries.

Sprinkle identifies as a sex-positive feminist, and much of her Activist and sex education work reflects this philosophy. In 2009, she appeared in the French documentary film Mutantes: Punk, Porn, Feminism, speaking about the beginnings of the movement as well as her own contributions to it.

In 2017, Sprinkle and her wife/collaborator Beth Stephens were official artists in Documenta 14, widely considered to be the most significant art exhibition in the world. They presented performances and visual art, lectured, and previewed their new film documentary, Water Makes Us Wet: An Ecosexual Adventure.