Arsenic trioxide (As₂O₃) increased human immunodeficiency virus type 1 (HIV-1) infectivity when particular Homo sapiens and Cercopithecus aethiops cell lines were used as targets. Knockdown of human TRIM5α by RNA interference eliminated the As₂O₃ effect, demonstrating that the drug acts by modulating the activity of this retroviral restriction factor. In contrast, HIV-1 infectivity in target cell lines from other primate species (Cercopithecus tantalus, Macaca mulatta, and Aotus trivirgatus) was not increased by As₂O₃, despite the potent TRIM5-dependent HIV-1 restriction activity that these cells exhibit. To determine if As₂O₃ responsiveness is characteristic of particular TRIM5 orthologues and not others, TRIM5 cDNAs from these five primate species were transduced into cat fibroblasts, which lack endogenous HIV-1 restriction activity and, therefore, responsiveness to As₂O₃. In this context, the HIV-1 restriction activity conferred by all TRIM5 orthologues was largely eliminated by As₂O₃. The effect of As₂O₃ on HIV-1 restriction is thus shared by different TRIM5 orthologues but dependent on factors specific to the cell line in which TRIM5 is expressed.