© 2015, American Society for Microbiology. Epidemiological studies have demonstrated that herpes simplex virus 2 (HSV-2) infection significantly increases the risk ofHIV-1 acquisition, thereby contributing to the expanding HIV-1 epidemic. To investigate whether HSV-2 infection directly facilitatesmucosal HIV-1 acquisition, we used our transgenic hCD4/R5/cT1 mouse model which circumvents major entry and transcriptionblocks preventing murine HIV-1 infection by targeting transgenic expression of human CD4, CCR5, and cyclin T1genes to CD4+ T cells and myeloid-committed cells. Productive infection of mucosal leukocytes, predominantly CD4+ T cells,was detected in all hCD4/R5/cT1 mice intravaginally challenged with an HIV-1 infectious molecular clone, HIV-Du151.2env-NLuc, which expresses an env gene (C.Du151.2) cloned from an acute heterosexually infected woman and a NanoLuc luciferasereporter gene. Lower genital tract HIV-1 infection after HIV-Du151.2env-NLuc intravaginal challenge was increased ~4-fold inhCD4/R5/cT1 mice coinfected with HSV-2. Furthermore, HIV-1 dissemination to draining lymph nodes was detected only inHSV-2-coinfected mice. HSV-2 infection stimulated local infiltration and activation of CD4+ T cells and dendritic cells, likelycontributing to the enhanced HIV-1 infection and dissemination in HSV-2-coinfected mice. We then used this model to demonstratethat a novel gel containing tenofovir disoproxil fumarate (TDF), the more potent prodrug of tenofovir (TFV), but not theTFV microbicide gel utilized in the recent CAPRISA 004, VOICE (Vaginal and Oral Interventions to Control the Epidemic), andFACTS 001 clinical trials, was effective as preexposure prophylaxis (PrEP) to completely prevent vaginal HIV-1 infection in almosthalf of HSV-2-coinfected mice. These results also support utilization of hCD4/R5/cT1 mice as a highly reproducible immunocompetentpreclinical model to evaluate HIV-1 acquisition across the female genital tract.