Objective To evaluate the effect of bile on endothelial nitric oxide synthase(NOS) activity. Methods Endothelial cells explanted from human umbilical vein were cultured in vitro, then bile of different concentrations 5%,10%,15%,20%,25% were mixed with the standard culture medium. Viability and morphology of the endothelial cells were studied, and NO(nitric oxide) content in conditioned medium and NOS activity were assessed. Results Viability and morphology of endothelial cells in the culture mediums presented as 5%,10%,15% bile were similar to the cells without bile medium while endothelial cells in mediums presented with 20% and 25% bile were markedly reduced in number and immature. Endothelial cell NOS activities in mediums presented with various concentration of bile were statistically decreased comparing with the cells in medium without bile. The quantity of NO in conditioned culture mediums with or without bile did not reach a statistical significance. Conclusion Bile appears to inhibit endotholial cells growth as well as endothelial NOS activity.