Nuclear factor kappa B (NF-κB) is a redox-associated transcription factor that is involved in the activation of survival pathways. We have previously shown that deoxycholate (DOC) activates NF-κB in hepatocytes and colon epithelial cells and that persistent exposure of HCT-116 cells to increasing concentrations of DOC results in the constitutive activation of NF-κB, which is associated with the development of apoptosis resistance. The mechanisms by which DOC activates NF-κB in colon epithelial cells, and whether natural antioxidants can reduce DOC-induced NF-κB activation, however, are not known. Also, it is not known if DOC can generate reactive oxygen species within mitochondria as a possible pathway of stress-related NF-κB activation. Since we have previously shown that DOC activates the NF-κB stress-response pathway in HCT-116 cells, we used this cell line to further explore the mechanisms of NF-κB activation. We found that DOC induces mitochondrial oxidative stress and activates NF-κB in HCT-116 cells through multiple mechanisms involving NAD(P)H oxidase, Na ⁺ /K ⁺ -ATPase, cytochrome P450, Ca ⁺⁺ and the terminal mitochondrial respiratory complex IV. DOC-induced NF-κB activation was significantly ( P < 0.05) inhibited by pre-treatment of cells with CAPE, EGCG, TMS, DPI, NaN ₃ , EGTA, Ouabain and RuR. The NF-κB-activating pathways, induced by the dietary-related endogenous detergent DOC, provide mechanisms for promotion of colon cancer and identify possible new targets for chemoprevention.