The TB9.8 of Mycobacterium bovis can induce strong antigen-specific T-cell responses in proliferation assays and IFN-γ assays. However, whether and how TB9.8 activates innate immune cells remain unclear. Therefore, recombinant protein TB9.8 (rTB9.8)-induced proinflammatory cytokine profile by RAW264.7 cells was investigated and the related signaling pathway was studied. Stimulation with rTB9.8 triggered RAW264.7 cells to produce IL-6 and IL-12 p40. In addition, rTB9.8 activated the mitogen-activated protein kinase (MAPK) cascade in RAW264.7 cells by inducing the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase (p38) and also promoted nuclear translocation of phosphorylated p38 and ERK1/2. Furthermore, rTB9.8 activated nuclear factor κB (NF-κB) signaling pathway by inducing p65 translocation into the nucleus and the phosphorylation of IκBα in the cytosol. Blocking assays showed that specific inhibitors of ERK1/2, p38, and IκBα can significantly reduce the expression of IL-6 and IL-12 p40, which demonstrated that rTB9.8-mediated cytokine production is dependent on the activation of these kinases. Thus, this study demonstrates that rTB9.8 can activate RAW264.7 and trigger IL-6 and IL-12 p40 production via the ERK, p38, and NF-κB signaling pathways.