Most of HIV-1 infections are acquired through sexual contact. In the absence of a preventive vaccine, the development of topical microbicides that can block infection at the mucosal tissues is needed. Dermaseptin S4 (DS4) is an antimicrobial peptide derived from amphibian skin, which displays a broad spectrum of activity against bacteria, yeast, filamentous fungi, and herpes simplex virus type 1. We show here that DS4 inhibits cell-free and cell-associated HIV-1 infection of P4-CCR5 indicator cells and human primary T lymphocytes. The peptide is effective against R5 and X4 primary isolates and laboratory-adapted strains of HIV-1. Its activity is directed against HIV-1 particles by disrupting the virion integrity. Increasing the number of DS4-positive charges reduced cytotoxicity without affecting the antiviral activity. The modified DS4 inhibited HIV-1 capture by dendritic cells and subsequent transmission to CD4(+) T cells, as well as HIV-1 binding on HEC-1 endometrial cells and transcytosis through a tight epithelial monolayer.