Human TRIM5α (TRIM5α_hu) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIV_mac). Alteration of arginine 332 in the TRIM5α_hu B30.2 domain to proline, the residue found in rhesus monkey TRIM5α, has been shown to create a potent restricting factor for both HIV-1 and SIV_mac. Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5α_hu. The increase in restricting activity correlated with an increase in the ability of TRIM5α_hu mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5α_hu R332P binding and allowed escape from restriction. The ability of TRIM5α_hu to restrict SIV_mac could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5α_hu B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5α_hu might potentiate the innate resistance of human cells to HIV-1 infection.