Interferon-mediated cellular antiviral responses are a key part of the mammalian innate immune system. Species-specific components of such responses, such as the interferon stimulated gene (ISG) repertoire, can contribute to the restriction of viral tropism to particular mammalian hosts. The picornavirus foot-and-mouth disease virus (FMDV) circulates naturally in multiple species of domestic livestock and wild animals, causing disease ranging from sub-clinical to fatal. This host range is thought to be limited to even-toed ungulates (cloven hoofed animals) while other mammalian species, including humans and equines, are not susceptible to disease and not implicated in the epidemiology of FMD. No basis for this host range has been described. In this study, screening of human and macaque ISG overexpression libraries identified primate proteins which act as restriction factors for FMDV infection of mammalian cultured cells. Comparative genomics revealed that the genes encoding some of these restriction factors exist only in species non-susceptible to FMD. Overexpression of these individual primate restriction factors in an otherwise highly susceptible porcine cell line rendered the culture refractory to FMDV infection. Furthermore, targeting these endogenous restriction factors by siRNA knock down of protein expression made a human cell line more permissive for growth of FMDV. Overexpression of the FMDV restriction factors did not affect the human picornavirus EV-71, suggesting that different viruses in the picornavirus family have co-evolved with specific host species. Together, these data provide a novel mechanism for species-level tropism of FMDV.