Endotoxin (lipopolysaccharide, LPS)-induced tumor necrosis factor-α (TNF-α) release from Kupffer cells is critically involved in the pathogenesis of alcohol-induced liver injury. We recently reported that inhibition of alcohol-induced plasma endotoxin elevation contributes to the protective action of zinc against alcoholic hepatotoxicity. The present study was undertaken to determine whether zinc interferes with the endotoxin-TNF-α signaling pathway, and possible mechanism(s) by which zinc modulates the endotoxin-TNF-α signaling. Administration of LPS to metallothionein (MT)-knockout (MT-KO) mice and 129/Sv wild-type (WT) controls at 4 mg/kg induced hepatic TNF-α elevation at 1.5 hours, followed by liver injury at 3 hours. Zinc pretreatment (two doses at 5 mg/kg) attenuated TNF-α production and liver injury in both MT-KO and WT mice, indicating a MT-independent action of zinc. Immunohistochemical detection of the phosphorylation of I-κB and nuclear factor (NF)-κB in the liver of MT-KO mice demonstrated that zinc pretreatment abrogated LPS-induced NF-κB activation in the Kupffer cells. Fluorescent microscopy of superoxide by dihydroethidine and of zinc ions by Zinquin in the liver of MT-KO mice showed that zinc pretreatment increased the intracellular labile zinc ions and inhibited LPS-induced superoxide generation. These results demonstrate that zinc inhibits LPS-induced hepatic TNF-α production through abrogation of oxidative stress-sensitive NF-κB pathway, and the action of zinc is independent of MT. Thus, zinc may be beneficial in the treatment of LPS-induced liver injuries, such as sepsis and alcoholism.