Abstract Depot medroxyprogesterone acetate (Depo-Provera) has been associated with an increased risk of HIV acquisition. In a longitudinal study, we investigated the impact of Depo-Provera use by healthy women on expression of immune markers for HIV preference and on HIV infection ex vivo at baseline (visit 1), 1 mo (visit 2), and 3 mo (visit 3) after Depo-Provera treatment. We found a significant increase in the frequency and expression of integrin α4β7 on CD4+ T cells at visit 2. Interestingly, Hispanic but not black women exhibited a significant increase in integrin α4β7 cell numbers and expression levels at visit 2, whereas black but not Hispanic women exhibited a significant change in CCR5 and CD38 expression levels between visit 2 and visit 3. The frequency of terminal effector memory CD4+ T cells was decreased significantly in black women from visit 1 to visit 3. Virus production following ex vivo HIV infection of PBMCs was increased at visit 3 compared with visit 1. In black women, the frequency of HIV p24+CD4+ T cells was higher at visit 3 than at visit 1. Expression of integrin α4β7 on HIV p24+CD4+ T cells following ex vivo infection at visit 2 was significantly less than at visit 1. These results demonstrate that Depo-Provera alters the immune profile of peripheral CD4+ T cells and increases susceptibility to HIV infection ex vivo. The observation that these effects differed between women of different ethnicities has implications for developing effective and targeted strategies for HIV prevention.