The p66^Shc protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66^Shc in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66^Shc, with Ser³⁶ replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H₂O₂ increased reactive oxygen species and phosphorylation of p66^Shc on Ser³⁶ in HepG2 cells. Overexpression of p66^Shc promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H₂O₂. p66^Shc activation also resulted in increased Erk-1/2, Akt and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66^Shc phosphorylation on Ser³⁶. Increased p66Shc expression was associated with reduced mRNA levels of anti-oxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66^Shc Ala³⁶ mutant inhibited p66^Shc signaling, enhanced anti-oxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66^Shc protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared to control subjects. Conclusions: in human alcoholic steatohepatitis, increased hepatocyte p66Shc protein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing anti-oxidant pathways.