The transcription factor T-bet regulates the differentiation of CD4(+) T-helper type 1 (Th1) cells and represses Th2 lineage commitment. Since Th1 cells are crucial in the defense against pathogens, several studies addressed the role of T-bet in immunity to infection using T-bet knockout (T-bet(-/-)) mice. Nevertheless, it is still unclear whether T-bet is required for defense. Although vaccinia virus (VV) has extensively been used as an expression vector and the smallpox vaccine, there is only limited knowledge about immunity to VV infection. The urgency to understand the immune responses has been increased because of concerns about bioterrorism. Here, we show that T-bet is critical in the defense against VV infection as follows: (i) the survival rate of T-bet(-/-) mice was lower than that of control littermates postinfection; (ii) T-bet(-/-) mice lost more weight postinfection; and (iii) control mice cleared VV faster than T-bet(-/-) mice. As expected, a significant Th2 shift was observed in CD4(+) T cells of T-bet(-/-) mice. Furthermore, absence of T-bet impaired VV-specific CD8(+) cytotoxic T-lymphocyte (CTL) function, including cytolytic activity, antiviral cytokine production, and proliferation. Cytolytic capacity of natural killer (NK) cells was also diminished in T-bet(-/-) mice, whereas anti-VV antibody production was not impaired. These data reveal that the enhanced susceptibility to VV infection in T-bet(-/-) mice was at least partially due to the Th2 shift of CD4(+) T cells and the diminished function of VV-specific CTLs and NK cells but not due to downregulation of antibody production.