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Oral tenofovir-based pre-exposure prophylaxis (PrEP) is an important tool for prevention of new HIV infections, which also reduces subclinical herpes simplex virus type 2 (HSV-2) shedding and symptomatic lesions in HIV-negative, HSV-2-seropositive individuals. However, the impact of PrEP on mucosal immunity has not been examined in detail.Here we evaluate paired genital tissue and systemic immune profiles to characterize the immunological effects of PrEP in HIV-negative, HSV-2-seropositive African women sexually exposed to HIV.We compared local and systemic innate and T-cell characteristics in samples collected during PrEP usage and 2 months after PrEP discontinuation.We found that frequencies of cervical CCR5+CD4+ cells, regulatory T cells, and tissue macrophages were significantly reduced during PrEP use compared with after PrEP discontinuation. In contrast, peripheral blood CD4+ and CD8+ T cells expressing markers of activation and trafficking were increased during PrEP usage.Together, our data are consistent with PrEP altering immunity differentially in the female genital tract compared with circulation in HSV-2+ women. Further study including comparison with HSV-2 negative women is needed to define the overall impact and mechanisms underlying these effects. These results point to the critical need to study the human mucosal compartment to characterize immune responses to mucosal infections.