In the event of smallpox bioterrorism, widespread vaccination may be required. Vaccinia immune globulin (VIG) has been used to treat complications from the smallpox vaccine. While the potency of VIG was defined by its ability to neutralize intracellular mature virus, a second form of vaccinia called the extracellular enveloped virus (EEV) is critical for virus spread in the host. The B5R-protein is one of many EEV-specific proteins. Immunoprecipitation and ELISA revealed that VIG recognizes the B5R-protein. An EEV plaque-reduction assay using a recombinant vaccinia that lacks the majority of the extracellular domain of B5R showed that the ability of VIG to neutralize EEV is principally directed at B5R. In addition, absorbing out the anti-B5R antibody present in VIG through the addition of recombinant B5R protein abrogated VIG's ability to significantly neutralize wild-type EEV. This work demonstrates the prominent role of B5R as a target of EEV-neutralizing activity of human antibodies.