Vaccinia-immune globulin (VIG) was used to treat severe complications of smallpox vaccination, but its use was controversial because it resolved disease in only some clinical cases. VIG is a pool of hyperimmune sera collected from individuals with a high neutralizing titre against the intracellular mature form (IMV) of vaccinia virus (VACV), but activity against the extracellular enveloped form (EEV) was often not considered. Here, the efficacy of anti-VACV antibodies (Abs) in protecting mice from intranasal infection with the VACV strain Western Reserve (WR) was evaluated. Mice were immunized passively with hyperimmune rabbit Abs (IgG) generated against inactivated IMV or produced following infection by VACV; subsequently, animals were challenged with VACV WR. The results demonstrated that: (i) good protection requires Abs to EEV in addition to IMV; (ii) Abs were effective when given before or up to 4 days after infection; and (iii) protection of mice from VACV WR correlated with a reduction of virus replication in lungs, but not in brain. In agreement with studies conducted before smallpox was eradicated and recent studies using EEV antigens for immunization, this study reiterates the importance of anti-EEV Abs in protecting against orthopoxvirus infection and illustrates the need to evaluate both anti-IMV and anti-EEV neutralizing Abs in VIG.