The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy is a topical microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal small inhibitory RNA (siRNA) application. To overcome the challenges of using siRNAs to knock down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in both the genital and rectal tracts of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity, provide durable target gene silencing for up to three weeks in vitro, and maintain effectiveness in a hydroxyethyl cellulose (HEC) gel formulation. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in cervicovaginal explants. When applied intravaginally to humanized mice, CD4-AsiCs provided durable protection against transmission of the virus. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent the sexual transmission of HIV.