Aim To investigate the effect of chlorpyrifos on relaxation function of artery and explore the potential mechanism. Methods Organ baths in basilar aortic rings of New Zealand rabbits were used to investigate the effect of different concentration chlorpyrifos on vascular relaxation function induced by acetylcholine(Ach) and sodium nitroprusside(SNP). The level of nitric oxide(NO) was measured in basilar artery and cultural human umbilical vein endothelial cells.The expression of endothelial nitricoxide synthase(eNOS) was detected by Real-time PCR and Western blot in cultural human umbilical vein endothelial cells(HUVEC). Results Endothelium dependent relaxation and NO content of rabbits basilar aortic rings were decreased in concentration-dependent manner by pre-treatment with chlorpyrifos(0.001 mmol/L,0.01 mmol/L,0.1 mmol/L) for 30 minutes. But chlorpyrifos had no effect on SNP-induced endothelium-independent relaxation of basilar aortic rings.Pre-treatment with atropine had no influence on the action of chlorpyrifos.The level of NO in culture medium and the expression of eNOS in HUVEC were decreased by pre-treatment with chlorpyrifos(0.001 mmol/L,0.01 mmol/L,0.1 mmol/L) for 30 minutes in concentration-dependent manner. Conclusion Chlorpyrifos decreases endothelium dependent relaxation of basilar aortic rings,which the mechanisms may relate to down-regulating of eNOS and decrease of NO induced by chlorpyrifos.