The transforming growth factor β (TGF-β) pathway represents an important signaling pathway involved in the regulation of diverse biological processes, including cell proliferation, differentiation, and apoptosis. Despite the known role of TGF-βR-mediated signaling in suppressing immune response, its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here, we show for the first time that TGF-βR-Smad3/4 signaling pathway acts as a positive regulator for TLR2 induction by bacterium nontypeable Hemophilus influenzae (NTHi) in vitro and in vivo. The positive regulation of TLR2 induction by TGF-βR is mediated via a dual mechanism involving distinct signaling pathways. One mechanism involves functional cooperation between the TGF-βR-Smad3/4 pathway and NF-κB pathway. Another involves MAP kinase phosphatase 1 (MKP-1)-dependent inhibition of p38 MAPK, a known negative regulator for TLR2 induction. Moreover, we showed that TβR-mediated signaling is probably activated by NTHi-derived TGF-β mimicry molecule via an autocrine-independent mechanism. Thus, our study provides new insights into the role of TGF-β signaling in positively regulating host defense response by tightly controlling the expression level of TLR2 during bacterial infections and may lead to new therapeutic strategies for modulating host defense and inflammatory response.