The 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis is strongly correlated with subversion of innate immune responses against invading mycobacteria. To understand the role of ESAT-6 in macrophage response against M. tuberculosis, the effects of ESAT-6 on macrophage generation of reactive oxygen species (ROS) and production of cytokines were studied. ESAT-6-induced macrophage secretion of monocyte chemoattractant protein-1 and TNF-α was found in a time- and dose-dependent manner. Signaling inhibition experiments indicate that NF-κB activation mediated by p38/JNK mitogen-activated protein kinase (MAPK) was involved in ESAT-6-triggered cytokine production. Moreover, TLR2 was engaged in ESAT-6-stimulated macrophage activation via rapidly induced ROS production and regulated activation of JNK/p38 MAPKs and NF-κB. More importantly, NADPH oxidase-mediated ROS generation is required during this process. Our study has identified a novel signal transduction pathway involving NADPH-ROS-JNK/p38-NF-κB in ESAT-6-induced cytokine production from macrophages. These findings provide an important evidence to understand the pathogenesis of M. tuberculosis infection in the modulation of the immune response.