Calderone was born in New York, New York on July 1, 1904. Biographer Jeffrey Moran suggests that her bohemian childhood (her father, Edward Steichen, was a noted photographer; her uncle was poet Carl Sandburg) and Quaker upbringing influenced her liberal outlook on sex as well as contributed to her opinionated and passionate nature. When Calderone was six, for instance, she berated the family-friend and sculptor Constantin Brâncuși for his horizontal-headed bird pieces, which would undoubtedly hinder the bird from singing. Brâncuși complied and began sculpting birds with more upturned heads.

Calderone attended the Brearley School in New York City for her secondary education. After graduation, she entered Vassar College, graduating in 1925 with an A.B. in Chemistry. Calderone decided to go into theatre after graduation and studied for three years at the American Laboratory Theater. She was also the model for the figures on the Pratt Institute flagpole, whose bronze was sculpted by her uncle Willard Dryden Paddock, which was erected in 1926 to commemorate the soldiers who served in World War I.

She abandoned acting and divorced in 1933. The death of her eight-year-old daughter Nell, along with dashed acting dreams and a divorce, plunged Calderone into depression. After a series of psychoanalytic tests, she decided to return to school and study medicine. She was 30 years old.

She obtained her M.D. degree from the University of Rochester medical school in 1939. She then received her M.P.H. from Columbia University in 1942. During this time she interned at hospitals and clinics, one belonging to Dr. Frank A. Calderone, whom she married in 1941. Frank Calderone was then a district health officer in New York and eventually became the chief administrative officer of the World Health Organization. Mary Calderone worked as a physician in the Great Neck, New York public school system. The couple had two daughters, Francesca (1943) and Maria (1946).

In 1953, Calderone joined the staff of the controversial Planned Parenthood Federation of America as its Medical Director. Her tenure there was prolific. In 1958, she organized a national conference that instigated the movement to legalize abortion. Her biggest success at Planned Parenthood came in 1964 when she overturned the American Medical Association policy against physicians disseminating information on birth control. Calderone did not believe that her work should be limited to preventive measures against pregnancy. Letters arrived at Planned Parenthood daily asking questions about not just sex, but sexuality at large. Calderone came to the realization that sexuality did not just equate genitality, and that sex education was sorely lacking from American society.

With the conviction that “handing out contraceptives was not enough,” Calderone quit her position at Planned Parenthood in 1964 and established the Sex Information and Education Council of the United States, Inc. (SIECUS). Driven by Calderone’s dynamic talks across the nation and its mission statement, “to establish man’s sexuality as a health entity,” the organization became an essential umbrella group for school administrators, sex educators, physicians, social activists, and parents seeking to access information about teaching sexuality education. Calderone and her organization became recognized and respected with the message of sex as a positive force, but opponents also watched her closely. Calderone’s insistence that sex education should begin as early as kindergarten did not impress religious conservative groups like MOMS (Mothers Organized for Moral Stability) and MOTOREDE (Movement to Restore Decency), who called Calderone the leader of the “SIECUS stinkpot.” A bestselling 1968 pamphlet, Is the School House the Proper Place to Teach Raw Sex?, targeted SIECUS, calling Calderone the “SIECUS Sexpot” and claiming that she wanted to undermine Christian morality and corrupt children.

By 1969, Calderone’s influence had been weakened by these attacks, and she stepped down as President, although she remained the Executive Director of SIECUS. Calderone published a rebuttal of the conservative attacks in the Vassar Quarterly, but according to Moran, it was a movement spearheaded by Playboy that would effectively fight the charges against sex education. Nevertheless, Calderone’s crusade for sexuality education with a “positive approach and moral neutrality” continued. Until 1982 she still held leadership positions at SIECUS and continued to expand sex education as a means to talk about other topics besides the sexual act, e.g. sexism, homosexuality, etc. Calderone widely gave talks, two of them at Vassar; her 1983 lecture as President’s Distinguished Visitor was titled “Sexuality in Infancy and Childhood—The Need for a Learning Theory.” She wrote several books on sex education: The Family Book about Sexuality and Talking with Your Child About Sex are two. Although Calderone was adamant about sexual freedom, her beliefs did not align with the burgeoning sexual revolution of the late 1960s. Calderone believed that the sex act should be ultimately reserved for marriage, and that sexuality found its peak expression through the “permanent man-woman bond.” In an article in Penthouse (magazine), and later in his book Sex By Prescription, the American psychiatrist Thomas Szasz criticized Calderone for her advocacy of the medicalization of sex, and her alleged hostility to homosexuals. Szasz described her as “confused and hypocritical” for telling an interviewer that she was “not suggesting the distribution of…contraceptive information to teenagers.”

In 1974, the American Humanist Association named her Humanist of the Year. Calderone was inducted into the National Women’s Hall of Fame in 1998.

Calderone died on October 24, 1998 at the Longwood Nursing Home in Kennett Square, Pennsylvania. She was 94.