CD38 is a progression marker in HIV-1 infection, it displays lateral association with CD4, and down-modulates gp120/CD4 binding. The aim of this study was to elucidate the mechanism behind the interplay between CD4, CD38, and HIV-1. We used mouse cell transfectants expressing human CD4 and either CD38 or other CD4-associated molecules to show that CD38 specifically inhibits gp120/CD4 binding. Human cell transfectants expressing truncated forms of CD38 and bioinformatic analysis were used to map the anti-HIV activity and show that it is concentrated in the membrane-proximal region. This region displayed significant sequence-similarity with the V3 loop of the HIV-1 gp120 glycoprotein. In line with this similarity, synthetic soluble peptides derived from this region reproduced the anti-HIV effects of full-length CD38 and inhibited HIV-1 and HIV-2 primary isolates from different subtypes and with different coreceptor use. A multiple-branched peptide construct presenting part of the sequence of the V3-like region potently and selectively inhibited HIV-1 replication in the nanomolar range. Conversely, a deletion in the V3-like region abrogated the anti-HIV-1 activity of CD38 and its lateral association with CD4. These findings may provide new insights into the early events of HIV-1 fusion and strategies to intervene.