Abstract: AIM To study the effects of cholestane-33β-5α-6β-triol on bovine aortic endothelial cells. METHODS The viability of endothelial cells was assessed by MTT assay. The concentration of malondialdehyde (MDA) in endothelial cells was measured by thiobarbituric acid (TBA) method. Apoptosis of endothelial cells was determined by flow cytometry and end-labeling in situ. RESULTS Cholestane-33β-5α-6β-triol (20-100 μmol·L^-1) decreased the endothelial cell viability rate in a time- and concentration-dependent manner. Cholestane-33β-5α-6β-triol (20 μmol·L^-1 and 100 μmol·L^-1) increased the content of MDA by 22 45% and 60.20% respectively at 18 h compared with cholesterol. Cholestane-33β-5α-6β-triol (100 μmol·L^-1 and 200 μmol·L^-1)induced apoptosis of endothelial cells, and the increase of apoptotic cells reached 12.73% and 18.80%, respectively. Otherwise, in the same condition, cholesterol (20-200 μmol·L^-1) showed no marked effect on endothelial cells. CONCLUSION Cholestane-3β-5α-6β-triol induced injury to endothelial cells by increasing the MDA content, decreasing the viability rate of endothelial cells and inducing apotosis of the endothelial cells.