Species-specific innate resistance against viral infections offers novel avenues for antiviral therapeutic and prophylactic approaches. The retroviral and lentiviral restriction factors Ref1 and Lv1 are variants of the tripartite motif protein TRIM5α, a component of cytoplasmic bodies. TRIM5α severely restricts productive retroviral infections at the postentry and preintegration steps by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5α_rh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5α_rh via a lentiviral vector in a permissive cell culture line, Magi-CXCR4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5α_rh transgene was stably introduced into CD34⁺ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5α_rh-expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5α_rh were also found to be phenotypically and functionally normal, expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHC II, and B7.1. These results demonstrate that the species-specific restriction factor TRIM5α_rh is effective in conferring HIV-1 resistance in a stem cell setting, thus paving the way for its application in AIDS gene therapy.