Homozygosity for the naturally occurring Δ32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted ∼50% of CCR5 alleles in a pool of primary human CD4⁺ T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4⁺ T cells had lower viral loads and higher CD4⁺ T-cell counts than mice engrafted with wild-type CD4⁺ T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4⁺ T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN–modified autologous CD4⁺ T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.