Targeting the mucosal immune system of the genital tract (GT) with subunit vaccines failed to induce potent and durable local immune response, crucial for protection against many sexually transmitted viral (STV) pathogens, including herpes simplex virus type 2 (HSV-2) that causes genital herpes. In this study, we investigated the potential of a novel lipopeptide/adenovirus type 5 (Lipo/rAdv5) prime/boost mucosal vaccine for induction of CD8+ T cell immunity to protect the female genital tract from herpes. The findings indicate that targeting the GT with a Lipo/rAdv5 prime/boost vaccine elicits a potent and long-lasting mucosal CD8+ T cell protective immunity against sexually transmitted herpes infection and disease.