Background: The use of progestin-only injectable contraception (IC) has been epidemiologically associated with increased risk of HIV acquisition but it remains unclear whether this relationship is causal and if so the mechanisms by which progestin-only contraceptives increase risk. Methods: Demographic and behavioral data along with blood and female genital tract (FGT) samples were obtained from a cohort of 18-23 year old HIV uninfected women in Durban South Africa. Cervical cytobrush cells were analyzed by flow cytometry and plasma progesterone measured by chemiluminescence. Cytokines in cervico-vaginal lavage (CVL) were measured by luminex. Results: Women using IC disproportionately accounted for new HIV infections during the study (RR 2.55 [1.90-3.42] p=0.0018). While 31.5% of women in the cohort used IC 72.7% of women who became HIV positive used IC (8 of 11); 9.09% of infected women did not use contraception. This difference was not accounted for by behavioral or demographic differences. The use of IC was significantly associated with a higher frequency of CD4+CCR5+ T cells of CD45+ cells in the cervix relative to those not on hormonal contraception (1.18% [0.33 4.53] vs. 0.29% [0.13 0.48] respectively; p=0.036). Women with high endogenous progesterone (>0.3ng/mL luteal phase) in the absence of IC use had significantly increased frequency of CD4+CCR5+ T cells in the cervix relative to those with low progesterone (follicular phase; 1.16% [0.51 3.76] of CD45+ cells vs 0.29% [0.13 0.48] respectively; p=0.042). High endogenous progesterone was also associated with a higher frequency of cervical CD4+CCR5+HLADR+CD38+ T cells (p=0.034). There was no observed difference based on IC use in soluble cytokine or chemokine levels in CVL. Conclusions: The increased frequency of HIV target cells at the site of exposure in the context of IC and high endogenous progesterone provides a potential biological mechanism for the increased HIV acquisition risk for women using injectable progestin-only contraceptives.