Macrophages are the primary host cells for Mycobacterium tuberculosis (Mtb). Although macrophages can mount a strong inflammatory response to dispose of invading microbial pathogens, the immune dysfunction of the Mtb-infected macrophage constitutes the hallmark of mycobacterial pathogenesis. A 10-kDa, Mtb secretory antigen (MTSA-10), encoded by ORF Rv3874, is one of the predominant members of the 'region of difference 1' locus of Mtb genome that has been strongly implicated in mycobacterial virulence. In this study, we investigated the possible role of MTSA-10 in modulating the macrophage dysfunction in a mouse macrophage cell line J774.1. We found that recombinant MTSA-10 caused extensive protein dephosphorylation in J774.1 cells as revealed by two-dimensional gel electrophoresis analysis. We also observed that MTSA-10 treatment downregulated the reactive oxygen species levels in the cells leading to activation of cellular protein phosphatases putatively responsible for the dephosphorylation phenomenon. This implied a direct role of MTSA-10 in the disruption of host cell signaling, resulting in downregulation of transcription of several genes essential for macrophage function.