Disruption of cyclophilin A (CypA)-capsid interactions affects HIV-1 replication in human lymphocytes. To understand this mechanism, we utilize human Jurkat cells, peripheral blood mononuclear cells (PBMCs), and CD4⁺ T cells. Our results show that inhibition of HIV-1 infection caused by disrupting CypA-capsid interactions is dependent on human tripartite motif 5α (TRIM5α_hu), showing that TRIM5α_hu restricts HIV-1 in CD4⁺ T cells. Accordingly, depletion of TRIM5α_hu in CD4⁺ T cells rescues HIV-1 that fail to interact with CypA, such as HIV-1-P90A. We found that TRIM5α_hu binds to the HIV-1 core. Disruption of CypA-capsid interactions fail to affect HIV-1-A92E/G94D infection, correlating with the loss of TRIM5α_hu binding to HIV-1-A92E/G94D cores. Disruption of CypA-capsid interactions in primary cells has a greater inhibitory effect on HIV-1 when compared to Jurkat cells. Consistent with TRIM5α restriction, disruption of CypA-capsid interactions in CD4⁺ T cells inhibits reverse transcription. Overall, our results reveal that CypA binding to the core protects HIV-1 from TRIM5α_hu restriction.