Vesicular stomatitis (VS) is a contagious and vector-borne disease of livestock caused by vesicular stomatitis virus (VSV). Vesicle formation in the oral mucosa, and in the skin of the coronary band (CB), teats, prepuce and vulva characterize the disease. Transmission occurs via direct animal-to-animal contact or via biting and non-biting insects. The pathogenesis is poorly understood. Viremia is rarely reported in livestock, viral replication kinetics in tissue is unknown, the role of insect bite in infection has not been established, and the detailed immune response to VSV acute infection has not been described. To address these questions steers were inoculated with VSV via scarification (SC) and black fly bite (FB) in the CB and in the skin of the neck. Animals were euthanized from 12 to 120 hours post infection (HPI). Multiple tissue samples and serum were collected for virus isolation and detection of numerous T helper 1 and 2 cytokines. Samples were also processed for immunohistochemistry to characterize the inflammatory cell infiltration. This study describes that VSV replicates to high copy numbers in specific anatomic regions, such as the CB, but fails to replicate and to cause lesions in the skin of the neck. Viral replication in the CB peaks between 24 and 48 HPI, with detection of replicating virus restricted to 24 HPI in the lymph nodes. Lesions are more prominent and develop faster in FB-inoculated animals. There is up-regulation of MHC II in SC-inoculated animals, with lack of MHC II up-regulation in the FB-inoculated animals, which may explain why lesions are more severe with FB. Marked T helper 1 cytokines are present at 48 HPI in the CB of infected animals, which comes immediately after the peak of virus replication. Strong and acute antiviral cytokine response may prevent the virus from systemic spread (viremia). Viral antigen presentation to B cell through follicular dendritic cells occurs as early as 72 HPI. This finding correlates with the fact that neutralizing antibodies are produced early in VSV infection. The results of this study add new data to better understand VS pathogenesis and might be instrumental for disease control and prevention.