Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5α-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5α orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5α-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5α on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5α does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5α. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5α and the putative CypA-regulated restriction factor.