The APJ receptor is widely expressed in the human central nervous system (CNS). Apelin was recently identified as the endogenous peptidic ligand for human APJ. Studies with animal models suggested that APJ and apelin play an important role in the hypothalamic regulation of water intake and the endocrine axis, in the regulation of blood pressure, and in cardiac contractility. Apelin has been found to block the activity of APJ as a human immunodeficiency virus type I (HIV-1) coreceptor. In this study, we combined chemical synthetic approaches with alanine substitution to evaluate the structural requirements for interactions with the APJ receptor. We demonstrated that apelin peptides in aqueous solution adopt a random conformation, and the positive charge and hydrophobic residues of apelin-13 play important roles in interactions with the APJ receptor. We have observed an important correlation between receptor binding affinity and cell−cell fusion inhibitory activity. The elucidation of structural requirements of apelin-13 in its interaction with the APJ receptor is critical for further investigation of apelin−APJ functions in vivo and in the design of small molecular inhibitors for potential treatment of HIV-1 infection in the CNS.