We demonstrate that Mycobacterium tuberculosis recombinant leucine-responsive regulatory protein (rLrp) inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), interleukin-6, and interleukin-12 production and blocks the nuclear translocation of subunits of the nuclear-receptor transcription factor NF-κB (Nuclear factor-kappa B). Moreover, rLrp attenuated LPS-induced DNA binding and NF-κB transcriptional activity, which was accompanied by the degradation of inhibitory IκBα and a consequent decrease in the nuclear translocation of the NF-κB p65 subunit. RLrp interfered with the LPS-induced clustering of TNF receptor-associated factor 6 and with interleukin-1 receptor-associated kinase 1 binding to TAK1. Furthermore, rLrp did not attenuate proinflammatory cytokines or the expression of CD86 and major histocompatibility complex class-II induced by interferon-gamma in the macrophages of Toll-like receptor 2 deletion (TLR2-/-) mice and in protein kinase b (Akt)-depleted mouse cells, indicating that the inhibitory effects of rLrp were dependent on TLR2-mediated activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt pathway. RLrp could also activate the PI3K/Akt pathway by stimulating the rapid phosphorylation of PI3K, Akt, and glycogen synthase kinase 3 beta in macrophages. In addition, 19 amino acid residues in the N-terminus of rLrp were determined to be important and required for the inhibitory effects mediated by TLR2. The inhibitory function of these 19 amino acids of rLrp raises the possibility that mimetic inhibitory peptides could be used to restrict innate immune responses in situations in which prolonged TLR signaling has deleterious effects. Our study offers new insight into the inhibitory mechanisms by which the TLR2-mediated PI3K/Akt pathway ensures the transient expression of potent inflammatory mediators.