Factors underlying HIV acquisition in women remain incompletely understood. This study evaluated ex vivo mucosal HIV-1_BaL infection (ectocervix, endocervix), T cell frequencies and phenotype (ectocervix, endocervix, peripheral blood), and HIV-1_BaL-induced tissue immune responses (ectocervix) in the proliferative and secretory phases of the menstrual cycle using samples obtained from women undergoing hysterectomies. Tissue infectivity (number of productively infected explants) and infection level following 500 and/or fifty 50% tissue culture infectious dose (TCID_50) HIV-1_BaL challenge were similar in the proliferative and secretory phases. Although not associated with infection outcomes, higher frequencies of HIV target CD4+α4β7+ T cells, and stronger HIV-1_BaL-induced proinflammatory responses were detected in ectocervix in the secretory versus proliferative phase. Independently of the cycle phase, serum E2 concentrations were inversely associated with ectocervical and endocervical tissue infection levels following high-dose 500 TCID_50 HIV-1_BaL challenge, with frequencies of CD4+α4β7+ T cells in endocervix, and with HIV-induced interleukin (IL)2R and IL4 in ectocervix. Although serum P4 concentrations and P4/E2 ratios were neither associated with tissue infection level nor infectivity, high P4 concentrations and/or P4/E2 ratios correlated with high frequencies of CD4+α4β7+ T cells in ectocervix, low frequencies of CD4+CD103+ blood T cells, low CD4+LFA-1+ T cells in endocervix, and high proinflammatory (IL1β, IL17, tumor necrosis factor α) ectocervical tissue responses to HIV-1_BaL. The data suggest an inhibitory effect of E2 on mucosal HIV infection, provide insights into potential mechanisms of E2-mediated anti-HIV activity, and highlight P4-associated immune changes in the mucosa.