There is considerable variability in host susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, but the host genetic determinants of that variability are not well understood. In addition to serving as a block for cross-species retroviral infection, TRIM5 was recently shown to play a central role in limiting primate immunodeficiency virus replication. We hypothesized that TRIM5 may also contribute to susceptibility to mucosal acquisition of simian immunodeficiency virus (SIV) in rhesus monkeys. We explored this hypothesis by establishing 3 cohorts of Indian-origin rhesus monkeys with different TRIM5 genotypes: homozygous restrictive, heterozygous permissive, and homozygous permissive. We then evaluated the effect of TRIM5 genotype on the penile transmission of SIVsmE660. We observed a significant effect of TRIM5 genotype on mucosal SIVsmE660 acquisition in that no SIV transmission occurred in monkeys with only restrictive TRIM5 alleles. In contrast, systemic SIV infections were initiated after preputial pocket exposures in monkeys that had at least one permissive TRIM5 allele. These data demonstrate that host genetic factors can play a critical role in restricting mucosal transmission of a primate immunodeficiency virus. In addition, we used our understanding of TRIM5 to establish a novel nonhuman primate penile transmission model for AIDS mucosal pathogenesis and vaccine research.