The possible bioterrorism threat using the variola virus, the causative agent of smallpox, has promoted us to further investigate the immunogenicity profiles of existing vaccines. Here, we study for the first time the immunogenicity profile of a replication-competent smallpox vaccine (vaccinia Tiantan, VTT strain) for inducing neutralizing antibodies (Nabs) through mucosal vaccination, which is noninvasive and has a critical implication for massive vaccination programs. Four different routes of vaccination were tested in parallel including intramuscular (i.m.), intranasal (i.n.), oral (i.o.), and subcutaneous (s.c.) inoculations in mice. We found that one time vaccination with an optimal dose of VTT was able to induce anti-VTT Nabs via each of the four routes. Higher levels of antiviral Nabs, however, were induced via the i.n. and i.o. inoculations when compared with the i.m. and s.c. routes. Moreover, the i.n. and i.o. vaccinations also induced higher sustained levels of Nabs overtime, which conferred better protections against homologous or alternating mucosal routes of viral challenges six months post vaccination. The VTT-induced immunity via all four routes, however, was partially effective against the intramuscular viral challenge. Our data have implications for understanding the potential application of mucosal smallpox vaccination and for developing VTT-based vaccines to overcome preexisting antivaccinia immunity.