Top of pageAbstract 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, TRIM5a, a component of cytoplasmic bodies. TRIM5a severely restricts productive retroviral infections at the level of post-entry and pre-integration by destabilizing the incoming viral capsid via ubiquitination. Using this approach, resistance to HIV-1 infection could be conferred by TRIM5arh expression in otherwise susceptible cells. Here we show that stable expression of simian TRIM5arh via a lentiviral vector in a permissive cell culture line, Magi-CXC4, conferred resistance to HIV-1. To translate these findings into a stem cell gene therapy setting, the TRIM5arh transgene was stable introduced into CD34+ hematopoietic progenitor cells to derive transgenic macrophages. Upon viral challenge, TRIM5arh expressing macrophages were highly resistant to HIV-1 infection compared to control cells. Human macrophages expressing TRIM5arh were also found to be phenotypically and functionally normal expressing the characteristic surface markers CD14, CD4, CCR5, CXCR4, MHCII, and B7.1. Based on these promising results, we sought to further improve the TRIM5a inhibiting action by designing more novel constructs by modifying the native human isoform of TRIM5a. These modified isoforms have also been shown to be effective in conferring viral resistance. These results demonstrate that the species specific restriction factor TRIM5arh is effective in conferring HIV-1 resistance in a stem cell setting thus paving the way for its application in AIDS gene therapy.