Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV).Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection.Vaccines are one of the most cost-effective medical treatments in modern civilization (80). A smallpox vaccine was the first human vaccine, and the modern smallpox vaccine, live vaccinia virus (VACV), is the most successful human vaccine, bringing about the worldwide eradication of smallpox disease due to a heroic World Health Organization campaign in the 1960s and 1970s (27). Elucidating the immunobiology underlying the protection provided by the smallpox vaccine will continue to reveal vaccinology principles that can be applied to future vaccine development against other infectious scourges (95). Neutralizing antibodies are of primary importance in the protection from smallpox provided by the smallpox vaccine in animal models (6,25,33,64) and humans (4). Vaccinia immune globulin (VIG) has been shown to be an effective treatment against smallpox (45) as it was able to reduce the number of smallpox cases by ϳ80% among exposed individuals in four case-controlled studies (42,45,50,51,66).Neutralizing antibodies confer protection mainly through the recognition of structures on the surface of virus particles, and therefore antiviral antibodies directed against the surface of virions are of primary inter...