In colonic epithelium, one of the pathways of lipopolysaccharide (LPS) activation of NF-κB and IL-8 is via Toll-like receptor (TLR)4, MyD88, IRAK1/4, and B-cell CLL/lymphoma 10 (Bcl10). However, this innate immune pathway accounts for only ∼50% of the NF-κB activation, so additional mechanisms to explain the LPS-induced effects are required. In this report, we identify a second pathway of LPS-induced stimulation, mediated by reactive oxygen species (ROS), in human colonic epithelial tissue cells in tissue culture and in ex vivo mouse colonic tissue. Measurements of IL-8, KC, Bcl10, phospho-IκBα, nuclear NF-κB, and phosphorylated Hsp27 were performed by ELISA. The TLR4-Bcl10 pathway was inhibited by Bcl10 siRNA and in studies with colonic tissue from the TLR4-deficient mouse. The ROS pathway was inhibited by Tempol, a free radical scavenger, or by okadaic acid, an inhibitor of Hsp27 dephosphorylation by protein phosphatase 2A (PP2A). The ROS pathway was unaffected in the TLR4-deficient tissue or by silencing of Bcl10. The combination of exposure to the free radical scavenger Tempol and of TLR4 or Bcl10 suppression was required to completely inhibit the LPS-induced activation. The ROS pathway was associated with dephosphorylation of Hsp27. LPS appears to activate both the regulatory component of the IκBα-kinase (IKK) signalosome through Bcl10 interaction with Nemo (IKKγ) and the catalytic component through Hsp27 interaction with IKKβ. Since LPS exposure is associated with septic shock and the systemic inflammatory response syndrome, distinguishing between these two pathways of LPS activation may facilitate new approaches to prevention and treatment.