Although vesicular stomatitis has been present for many years in the Americas, many aspects of its natural history remain undefined. In this study, we challenged five adult Virginia opossums (Didelphis marsupialis) with vesicular stomatitis New Jersey serotype virus (VSNJV). Opossums had no detectable antibodies against VSNJV prior to being inoculated with 10^6.5 median tissue culture infective doses (TCID₅₀) of VSNJV by two routes; intraepithelial/subepithelial (IE/SE) inoculation and scarification in the muzzle (SM). Clinical response was monitored daily and animals were tested for viral shedding. All infected animals developed vesicles and ulcers on the tongue and inflammation of the nasal alar folds. Virus was isolated from esophagus-pharynx, nasal, and from ocular swabs and lesions samples. The failure to detect viremia in these animals indicates that a source other than blood may be required for transmission to insect vectors. Our results suggest that D. marsupialis could play a role in the maintenance of VSNJV outside of domestic animal populations and could provide a model to study vesicular stomatitis virus pathogenesis.