Live-virus vaccines for smallpox are effective but have risks that are no longer acceptable for routine use in populations at minimal risk of infection. We have developed a mucosal, killed-vaccinia virus (VV) vaccine based on antimicrobial nanoemulsion (NE) of soybean oil and detergent. Incubation of VV with 10% NE for at least 60 min causes the complete disruption and inactivation of VV. Simple mixtures of NE and VV (Western Reserve serotype) (VV/NE) applied to the nares of mice resulted in both systemic and mucosal anti-VV immunity, virus-neutralizing antibodies, and Th1-biased cellular responses. Nasal vaccination with VV/NE vaccine produced protection against lethal infection equal to vaccination by scarification, with 100% survival after challenge with 77 times the 50% lethal dose of live VV. However, animals protected with VV/NE immunization did after virus challenge have clinical symptoms more extensive than animals vaccinated by scarification. VV/NE-based vaccines are highly immunogenic and induce protective mucosal and systemic immunity without the need for an inflammatory adjuvant or infection with live virus.