Hormones play an important role in the induction of protective immune responses after intravaginal (IVAG) immunization with an attenuated strain of herpes simplex type 2 (HSV-2 TK-). Previously we reported that ovariectomized (OVX) mice receiving estradiol (E) prior to immunization were not protected, while mice receiving progesterone (P) had sterile protection after IVAG HSV-2 challenge. Protection correlated with the ability of TK- to cause productive infection. Lack of protection in the E group was associated with the effect of E on thickening of vaginal epithelium. In the current study, we examined the effect of E on immunization after the thickening of vaginal epithelium subsided. OVX, C57BL/6 mice were given P, E or saline (S) for 3 days. The P and S groups were then immunized with IVAG TK-. To determine the time point at which E mice could be successfully immunized, TK- was given between days 1 and 7 (E1–E7) post-E treatment. Three weeks after immunization, mice were challenged with IVAG HSV-2. Protection was absent in E1–E4 groups, 100% in E6 and E7 and partially successful in E5. While the protection in P and S was sterile, protected mice in E5 and E6 groups had non-sterile protection. The presence of HSV-2 specific IgG antibodies in the vaginal washes after challenge closely correlated with sterile immunity. After challenge, vaginal associated lymphoid tissues (iVALTs) were seen in the vaginal tract of the protected groups, except for the E5 group. The cytokine environment after challenge was also examined. The results show that while immunization under the effect of E may lead to clearance of virus, the mechanism is quite different than that seen under other hormonal conditions. Local hormonal influences must be considered when designing vaccines for STDs.