The role of cholesterol in the pathogenesis of non-alcoholic steatohepatitis (NASH) remains unclear. It is known that apoptosis of hepatocytes is an important characteristics of NASH. The objective of this study was to investigate the effects of cholesterol on steatotic HepG2 cell apoptosis and the possible mechanism in vitro. In this study, HepG2 cells were divided into three groups: (1) normal group, (2) steatosis group and (3) cholesterol group. HepG2 cells were treated with oleic acid to establish a steatosis study model. Steatosis was assessed by Oil Red O staining and triglyceride content assay. Cell apoptosis was measured using an apoptosis kit. The expression levels of apoptosis-related proteins (P53, Bcl-2, Bax, caspase-3, cyclin A, cyclin B1 and cyclin E) were determined by western blot analyses. We found that a hepatocyte steatosis model was successfully established by oleic acid (200 mu mol/L) induction. The cholesterol (50 mg/L) group had similar amount of lipid droplets and triglyceride content as steatosis group (P > 0.5). However, the apoptosis rate (P < 0.01) of the cholesterol group was significantly higher than that of the normal group or the steatosis group, and the protein expressions of Bax and caspase-3, but not P53, Bcl-2, cyclin A, cyclin B1 and cyclin E, were also increased in the cholesterol group. Those results suggested that cholesterol markedly promoted apoptosis of steatosis HepG2 cells in vitro, likely through the up-regulation of Bax and caspase-3 expression. This study contributes to explain the effect of cholesterol on NASH pathogenesis.