Mycobacterium bovis is the etiological factor of bovine tuberculosis (BTB), posing a significant problem to domestic cattle. The bacterium is also zoonotic, affecting human health worldwide. Macrophage evasion of the bacterium involves mycobacterial molecules such as MB1684 (ornithine carbamoyltransferase). In this study, we confirmed a concentration-dependent decrease in proliferation of Ana-1 macrophages when treated with rMB1684 when compared with mycobacterium bovis purified protein derivative of tuberculosis (MbPPD) or phosphate buffer solution incubation groups. We examined the activation of nuclear factor-kappa B (NF-κB) upon exposure to MB1684, and its role in MB1684-induced upregulation of interferon (IFN)-γ and proinflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) in Ana-1 macrophages. The levels of proinflammatory cytokines and IFN-γ were significantly high in MB1684-treated Ana-1 macrophages. The treatment led to an increase in NF-κB activation and a high expression of the just mentioned proinflammatory cytokines. NF-κB inhibition significantly abrogated MB1684-induced upregulation of proinflammatory cytokine mRNA expression, which suggests that MB1684-induced activation of NF-κB in turn stimulates gene expression of IFN-γ and proinflammatory cytokines in Ana-1 macrophages. The experiment was repeated in bone marrow macrophages, a more in-vivo-like model system, and similar results validated our conclusion. Further, we identified the possibility of the application of MB1684 antigen for the detection of BTB in cattle serum.