Background: Depot-medroxyprogesterone acetate (DMPA) has been associated in some studies with increased HIV susceptibility in women. We used a pigtail macaque model to document the effects of repeated DMPA treatments and their potential contribution to increased SHIV susceptibility. Methods: Nine pigtails were administered 2.5, 1.5, or 0.5 mg/kg DMPA in study weeks one and four. Menstrual cycling, vaginal epithelial thickness, and other SHIV susceptibility factors were monitored for a mean of 24 study weeks. Results: All DMPA treatments suppressed menstrual cycling and increased vaginal pH. The vaginal epithelium thinned naturally during baseline menstrual cycles (from mean of 351 to 161 μm in late-luteal phase). Following DMPA, the non-nucleated layer was temporarily absent. Two weeks post-second DMPA injection, mean epithelial thickness was 53, 45, and 167 μm for the descending doses, respectively. Conclusions: All animals showed temporal vaginal epithelial thinning with loss of the non-nucleated layer, and vaginal pH changes post-DMPA injections.