We previously demonstrated that Mycobacterium tuberculosis (M. tbc)-induced interleukin (IL)-12 expression is negatively regulated by the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) 1/2 pathways in human monocyte-derived macrophages (MDMs). To extend these studies, we examined the nature of the involvement of toll-like receptors (TLRs) and intracellular signalling pathways downstream from PI3K in M. tbc-induced IL-23 expression in human MDMs. M. tbc-induced Akt activation and IL-23 expression were essentially dependent on TLR2. Blockade of the mammalian targets of rapamycin (mTOR)/70 kDa ribosomal S6 kinase 1 (S6K1) pathway by the specific inhibitor rapamycin greatly enhanced M. tbc-induced IL-12/IL-23 p40 (p40) and IL-23 p19 (p19) mRNA and IL-23 protein expression. In sharp contrast, p38 mitogen-activated protein kinase (MAPK) inhibition abrogated the p40 and p19 mRNA and IL-23 protein expression induced by M. tbc. Furthermore, the inhibition of PI3K-Akt, but not ERK 1/2 pathway, attenuated M. tbc-induced S6K1 phosphorylation, whereas PI3K inhibition enhanced p38 phosphorylation and apoptosis signal-regulating kinase 1 activity during exposure to M. tbc. Although the negative or positive regulation of IL-23 was not reversed by neutralization of IL-10, it was significantly modulated by blocking TLR2. Collectively, these findings provide new insight into the homeostatic mechanism controlling type 1 immune responses during mycobacterial infection involving the intracellular network of PI3K, S6K1, ERK 1/2 and p38 MAPK pathways in a TLR2-dependent manner.