The mechanisms by which steatosis renders hepatocytes susceptible to damage in non-alcoholic steatohepatitis (NASH) are unclear although fat accumulation is believed to increase hepatocyte susceptibility to inflammatory cytokines and oxidative stress. We therefore investigated the susceptibility of steatotic, hepatocyte-derived cells to TNFalpha and the pro-oxidant, t-butylhydroperoxide (TBH). HepG2 spheroids rendered steatotic by fat-loading with 0.15 mM oleic or palmitic acid for 48 h and treated with TNFalpha or TBH for 18 h exhibited surprisingly lower levels of cytotoxicity, and increased anti-oxidant activity (superoxide dismutase (SOD)) compared with non fat-loaded controls. The protective effect of steatosis was significantly reversed by the inhibition of AMP-activated kinase (AMPK) since spheroids transfected with a kinase-dead AMPKalpha2 subunit, exhibited a significant increase in TBH-induced cytotoxicity when fat-loaded. In conclusion, our findings suggest that fat-loaded hepatocyte-derived cells are surprisingly less susceptible to cytokine and pro-oxidant induced damage via an adaptive mechanism dependent, in part, on AMPK activity.