The natural transmission of vesicular stomatitis New Jersey virus (VSNJV), an arthropod-borne virus, is not completely understood. Rodents may have a role as reservoir or amplifying hosts. In this study, juvenile and nestling deer mice (Peromyscus maniculatus) were exposed to VSNJV-infected black fly (Simulium vittatum) bites followed by a second exposure to naive black flies on the nestling mice. Severe neurological signs were observed in some juvenile mice by 6 to 8 days postinoculation (DPI); viremia was not detected in 25 juvenile deer mice following exposure to VSNJV-infected fly bites. Both juvenile and nestling mice had lesions and viral antigen in the central nervous system (CNS); in juveniles, their distribution suggested that the sensory pathway was the most likely route to the CNS. In contrast, a hematogenous route was probably involved in nestling mice, since all of these mice developed viremia and had widespread antigen distribution in the CNS and other tissues on 2 DPI. VSNJV was recovered from naive flies that fed on viremic nestling mice. This is the first report of viremia in a potential natural host following infection with VSNJV via insect bite and conversely of an insect becoming infected with VSNJV by feeding on a viremic host. These results, along with histopathology and immunohistochemistry, show that nestling mice have widespread dissemination of VSNJV following VSNJV-infected black fly bite and are a potential reservoir or amplifying host for VSNJV.