The mucosal surface of the vagina is a primary human immunodeficiency virus (HIV) entry portal， making it an attractive site for HIV vaccination. However， HIV vaccines based on recombinant adenovirus (rAd) do not efficiently cross the mucus layers or underlying epithelium of the vagina. Here we designed nanocomplexes of rAd particles coated with (1) the polyethylene glycol derivative APS to provide a hydrophilic surface that would prevent entrapment in the hydrophobic mucus， and (2) the cell-penetrating peptide TAT to improve transduction efficiency. The optimized rAd-TAT-APS nanocomplexes could achieve the balance of effective mucus-penetrating and cellular transduction. Intravaginal delivery of rAd-TAT-APS encoding HIVgag p24 into mice strongly enhanced HIVgag-specific systemic and mucosal immune responses. This rAd-TAT-APS system may allow effective vaginal delivery of vaccines against HIV and other infectious agents.