Mammalian cells express several factors that act in a cell-autonomous manner to inhibit retrovirus replication. Among these are the Friend virus susceptibility factor 1/lentivirus susceptibility factor 1/restriction factor 1 (Ref1) class of restriction factors, which block infection by targeting the capsids of diverse retroviruses. Here we show that lentivirus susceptibility factor 1 and Ref1 are species-specific variants of tripartite interaction motif 5α (TRIM5α), a cytoplasmic body component recently shown to block HIV-1 infection in rhesus macaque cells, and can indeed block infection by widely divergent retroviruses. Depletion of TRIM5α from human cells relieved restriction of N-tropic murine leukemia virus (N-MLV), and expression of human TRIM5α in otherwise nonrestricting cells conferred specific resistance to N-MLV infection, indicating that TRIM5α is Ref1 or an essential component of Ref1. TRIM5α variants from humans, rhesus monkeys, and African green monkeys displayed different but overlapping restriction specificities that were quite accurately predicted by the restriction properties of the cells from which they were derived. All TRIM5α variants could inhibit infection by at least two different retroviruses, and African green monkey TRIM5α was able to inhibit infection by no less than four divergent retroviruses of human, non-human primate, equine, and murine origin. However, each TRIM5α variant was unable to restrict retroviruses isolated from the same species. These data indicate that TRIM5α can confer broad innate immunity to retrovirus infection in primate cells and is likely to be an important natural barrier to cross-species retrovirus transmission.