To study the role of c-Src kinase in pro-oxidant-induced stimulation of Toll-like receptor 4 (TLR4), we used lipopolysaccharide from Escherichia coli K12 (LPS-EK) and monophosphoryl lipid A, as TLR4-specific agonists and positive controls, and SIN-1 and potassium peroxychromate as pro-oxidant sources. We used the HEK-Blue mTLR4 cell line, which is stably transfected with mouse TLR4 and expresses optimized SEAP reporter under the control of a promoter inducible by NF-κB transcription factor. The level of SEAP released due to TLR4 stimulation was a measure of NF-κB activation. Treatment with either the pro-oxidants or LPS-EK increased SEAP release and TNF-α production in these cells. These treatments also increased intracellular reactive oxygen species accumulation, with an enhanced production of nitric oxide and TBARS to confirm oxidant stress in these cells. Pretreatment with c-Src kinase inhibitors, PP2 and Ca-pY, which act by different mechanisms, decreased these parameters. Pretreatment with SSG, a c-Src activator, enhanced the effects promoted by LPS-EK and pro-oxidants and rescued cells from the PP2- and Ca-pY-induced effects. Curiously, pro-oxidants, but not TLR4 agonist, increased the ratio of TNF-α to IL-10 released, suggesting that pro-oxidants can initiate and maintain an imbalance of TNF-α production over IL-10. To different degrees, both pro-oxidants and TLR4 agonist increased formation of c-Src complexes with TLR4 and IκB-α as coimmunoprecipitates. Both pro-oxidants and TLR4 agonist increased c-Src phosphorylation of the Tyr42 residue in IκB-α, but the pro-oxidant-induced effect was more robust and much longer lasting. Taken together, these studies provide a mechanism whereby c-Src assumes a central role in pro-oxidant-induced NF-κB activation in TLR4 signaling. Pro-oxidant-induced activation of TLR4 through c-Src/NF-κB/IκB-α coupling provides a basis for a molecular dissection of the initiation and maintenance of sterile inflammation that may serve as a “pathophysiologic primer” for many diseases.