Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2-/-) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1-/- mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2-/- mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2-/- mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2-/- mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2-/- mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2-/- mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2-/- mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon.