In addition to the CCR5 and CXCR4 chemokine receptors, a subset of primary human immunodeficiency virus type 1 (HIV-1) isolates can also use the seven-transmembrane-domain receptor APJ as a coreceptor. A previously identified ligand of APJ, apelin, specifically inhibited the entry of primary T-tropic and dualtropic HIV-1 isolates from different clades into cells expressing CD4 and APJ. Analysis of apelin analogues demonstrated that potent and specific antiviral activity was retained by a 13-residue, arginine-rich peptide. Antiviral potency was influenced by the integrity of methionine 75, which contributes to APJ-binding affinity, and by the retention of apelin residues 63 to 65. These studies demonstrate the ability of a small peptide ligand to block the function of APJ as an HIV-1 coreceptor, identify apelin sequences important for the inhibition, and provide new reagents for the investigation of the significance of APJ to HIV-1 infection and pathogenesis.