Human TRIM5alpha (TRIM5alpha(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 TRIM5alpha(hu) B30.2 domain to proline, the residue found in rhesus monkey TRIM5alpha, 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 TRIM5alpha(hu.) The increase in restricting activity correlated with an increase in the ability of TRIM5alpha(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 TRIM5alpha(hu) R332P binding and allowed escape from restriction. The ability of TRIM5alpha(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 TRIM5alpha(hu) B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5alpha(hu) might potentiate the innate resistance of human cells to HIV-1 infection.