The Mtb9.8 antigenic protein of Mycobacterium bovis/Mycobacterium tuberculosis has been identified as a target of the T-cell response. However, the interaction of Mtb9.8 with Toll-like receptors (TLRs) and the relevant signaling pathways have not been fully clarified. In this study, recombinant Mtb9.8 (rMtb9.8) derived from M. bovis-stimulated RAW264.7 cells initiated the secretion of TNF-α and IL-1β in a dose-dependent manner. Blocking assays show that TLR2-neutralizing antibody decreases the production of TNF-α and IL-1β. Moreover, NF-κB activation is associated with TNF-α and IL-1β production by rMtb9.8 stimulation, and rMtb9.8 stimulation also induces the phosphorylation of NF-κB p65 at Ser536 and its rapid nuclear translocation in RAW264.7 cells. Furthermore, NF-κB luciferase activity is rapidly activated in response to rMtb9.8 in RAW264.7 cells and is also significantly increased in rMtb9.8-induced HEK293-TLR2. However, these activations were abrogated in cells with a dominant-negative mutation of NF-κB p65 and by treatment with anti-TLR2 antibody. We also find that rMtb9.8 induces the activation of IRF-1. These findings indicate that M. bovis-derived rMtb9.8 activates the NF-κB pathway via TLR2 in RAW264.7 cells. In particular, it phosphorylates NF-κB p65 at Ser536 and induces nuclear translocation, thereby leading to the production of TNF-α and IL-1β, which correlates with the induction of IRF-1.