The goal of this study is to investigate the effects of glucose fluctuation on cultured human L02 hepatocytes in vitro and discuss its possible mechanism. Cultured cells were randomly divided into 4 groups: normal (N), high glucose (HG), glucose fluctuation (GF) and osmotic pressure (OP). After 72 hours, alanine transferase (ALT), aspartate amino transferase (AST) and lactate dehydrogenase (LDH) in the medium were measured. In the cells, glycogen, glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), Na+K+-ATP and Ca2+Mg2+-ATP enzyme activities were detected. In addition, cell membrane fluidity was also detected. Compared with the N group, the activities of ALT，AST and LDH in the HG and GF groups increased markedly, while the activities of GSH, SOD, Na+K+-ATP and Ca2+ Mg2+-ATP enzymes were decreased significantly. The contents of glycogen and MDA in the cells of HG and GF groups were increased obviously, while cell membrane fluidity was decreased markedly (all p＜0.01). There are also significantly statistical differences between the HG and GF groups(all p＜0.01). Our results demonstrate that glucose fluctuation can change the permeability of cell membrane, lead to the leakage of enzymes and cause marked oxidative damage and cell toxicity in human L02 hepatocytes. Moreover, the deleterious effects of GF on the L02 cells are significantly higher than that of the HG group.