Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), remains one of the most widespread and deadly diseases, accounting for nearly two million deaths annually. It is estimated that one third of the population worldwide is latently infected with Mtb, and the explosion of the HIV epidemic greatly increases the rate of active, infectious TB progression from latent TB. MPT63/Rv1926c and MPT64/Rv1980c, a major target of cell-mediated immunity in tuberculosis, is proposed to be a promising candidate for novel vaccines and diagnostic tests; it also involves in virulence mechanism of Mtb. In this study, we investigated the effect of MPT63 and MPT64 in toll-like receptor (TLR)s activation of murine macrophage, and explored the possible mechanism. Here we show that TLR ligands-induced innate immune responses was differential regulated by MPT63/Rv1926c and MPT64/Rv1980c in murine macrophages. Thus, these results provide novel insight into the crucial role of MPT in TLR signaling-mediated innate immune responses.