Vaccinia virus (VV) has been used extensively as a vaccine vehicle in the clinical application for infectious diseases and cancer. Previous studies have suggested that the unique potency of VV-based vaccine lies in its effective activation of the innate immune system. However, how VV activates innate immune pathways remains largely unknown. In this study, we showed that VV elicited innate immune response through both Toll-like receptor (TLR)-dependent and -independent pathways. The TLR pathway was mediated by TLR2 and MyD88, leading to the production of proinflammatory cytokines, whereas activation of the TLR-independent pathway resulted in the secretion of IFN-beta. More importantly, both TLR-dependent and -independent pathways were required for activating innate and adaptive immunity to VV in vivo. These findings represent the first evidence that innate immune recognition of VV is mediated by TLR2, demonstrate that one pathogen can target both TLR and non-TLR innate immune pathways to work together in achieving efficient activation of host defense, and suggest potential new strategies for the design of effective vaccines.