M ammalian and venom secreted phospholipases A₂ (sPLA₂s) have been associated with a variety of biological effects. Here we show that several sPLA₂s protect human primary blood leukocytes from the replication of various macrophage and T cell–tropic HIV-1 strains. Inhibition by sPLA₂s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA₂s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA₂s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA₂ have no effect on HIV-1 infection, suggesting that sPLA₂ catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA₂s to host cells. Indeed, of 11 sPLA₂s from venom and mammalian tissues assayed, 4 venom sPLA₂s were found to be very potent HIV-1 inhibitors (ID₅₀ < 1 nM) and also to bind specifically to host cells with high affinities (K_0.5 < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA₂s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA₂s are being characterized in humans.