This study aimed to evaluate the histologic and cytologic effects of preoperative vaginal estrogen in women with atrophic vaginitis and pelvic organ prolapse.Forty-two women with atrophic vaginitis and stage greater than or equal to 2 prolapse were enrolled in this assessor-blinded randomized controlled trial comparing daily vaginal estrogen cream use for 2 to 12 weeks preoperatively versus no intervention. Data were analyzed using t test and analysis of variance.Of these 42 women, 22 received treatment and 20 were controls. After a mean 7 (3) weeks of use, the vaginal maturity index increased 15.5% in the treatment group and declined 1.5% in the control group (P < 0.001). The vaginal epithelial thickness was 339 (96) μm in the treatment group compared to 302 (119) μm (P = 0.275) in the controls.Preoperative vaginal estrogen application for 2 to 12 weeks restores vaginal cytology to premenopausal levels, but does not increase vaginal epithelial thickness in women with prolapse.