The host range of human immunodeficiency virus type 1 (HIV-1) is extremely narrow, which has hampered the establishment of non-human primate models for HIV-1 infection. The species-specific innate immune factor tripartite motif 5 alpha (TRIM5α) is a key molecule that confers potent resistance against HIV-1 infection. In this study, we targeted the TRIM5α gene of rhesus macaques (rhTRIM5α) using the transcription activator-like effector nuclease (TALEN) to study the effect on HIV-1 infection. CD4+ T cells were separated from the peripheral blood of rhesus macaques by magnetic cell sorting, and the positive rate was greater than 99%. TALEN plasmids targeting rhTRIM5α were constructed and introduced into CD4+ T cells by electroporation, with a transfection efficiency of approximately 25%. The genome of the targeted cells was extracted, and the target efficiency was analyzed by T7E1 enzyme digestion. After sorting the positive transductants, the TALENs induced rhTRIM5α mutations at a rate of more than 40%. The ability of the HIV-1 virus to infect the targeted cells was demonstrated by ELISA. The results showed that targeting rhTRIM5α enhanced the susceptibility to HIV-1 infection. This finding will pave the way for further establishment of a new rhesus macaque model for HIV-1 studies.