Objective To explore the effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells and underlying mechanism. Methods The thoracic aorta of newborn calf was used for primary endothelial cells culture. Cells were divided into 3 groups and cultured for 48 h : control group ( C, 5.5 mmol/L), constant high glucose group ( HG, 30 mmol/L) and glucose fluctuation ( GF, three circles of 2 h 30 mmol/L followed by 3 h 5.5 mmol/L, 30 mmol/L overnight, repeat the whole procedure on the following day) groups. The membranes fluidity of endothelial cells was detected by fluorescence polarization method. The contents of sorbierite, aldose reductase (AR), sorbitoi dehydrogenase (SDH) and advanced glycation end products (AGEs) were measured. RAGE, eNOS and ET-1 mRNA expressions were detected by semi-quantitative RT-PCR. Results The membranes fluidity of endothelial cells in HG or GF group were significantly decreased compared with the control group( all P <0.01 ) and significantly lower in GF group than those in HG group (all P < 0. O1 ). Sorbierite, AR and AGEs concentrations were significantly higher in HG and GF groups than those in control group ( all P <0. 01 ) and AR and AGEs concentrations were significantly higher in GF group than that in HG group ( all P <0.01 ). SDH of endothelial cells in HG or GF group were decreased compared with the control group and lower in GF group than in HG group ( all P < 0.05). In addition, the mRNA levels of RAGE, eNOS and ET-1 were significantly upregulated compared with the control group( all P < 0.01 ). Conclusions Glucose concentration fluctuation can result in more severe bovine arterial endothelial cells dysfunction than high glucose via activating polyols metabolic pathways, upregulating the expression of AGEs, eNOS and ET-1.Therefore, glucose concentration fluctuation might play a crucial role on macrovascular complications of diabetes.