Endogenous transforming growth factor-beta1 (TGF-β1) plays an important role in the negative regulation of toll-like receptor (TLR) signaling in a feedback manner. Suppressors of cytokine signaling 3 (SOCS3) has been shown to be induced by TGF-β1 in osteoclast/macrophage, while the reports on the role of SOCS3 in regulating TLR4 signaling were controversial. The functional relationship between SOCS3 and TGF-β1/Smad3 pathway in TLR4 response also remains unclear. In this study, we demonstrate that LPS-induced endogenous TGF-β1 contributes to the inducible SOCS3 expression in macrophages. SOCS3 silencing could markedly decrease the LPS-induced production of TNF-α and IL-6 in macrophages. Interestingly, less decrease of LPS-induced TNF-α, IL-6 by SOCS3 silencing was observed in Smad3 null macrophages. Furthermore, we found SOCS3 could interact with Smad3, and inhibit Smad3 nuclear translocation and transcriptional activity. Therefore, our data demonstrate that SOCS3 is a positive regulator of TLR4 response by feedback inhibiting endogenous TGF-β1/Smad3 signaling, thus outlining a new feedback regulatory manner for TLR4 response in macrophages.