Aim To study the mechanisms of caveolin-1 （Cav-1） up-regulating the extracellular Ca^2＋-sensing receptor （CaR）-induced endothelial nitric oxide synthetase （eNOS） activation in human umbilical vein endothelial cells （HUVECs）. Methods Cultured HUVECs, the same generation of cells were randomly divided into: （1）control group （2）CaR agonist （spermine, 2.0 mmol/L）＋Ca^2＋ group （3）caveolae structural damage （filipin, 1.5 mg/L）＋spermine＋Ca^2＋ group （4）Cav-1 short hairpin RNA （Cav-1 ShRNA）＋spermine＋Ca^2＋ group （5）vehicle＋spermine＋Ca^2＋ group （6）different concentrations （1.5, 2.0, 2.5 mg/L） filipin groups. Western blotting experiments were performed to detect protein expression of Cav-1, eNOS, phosphorated eNOS （p-eNOS） and expressions of Cav-1 and eNOS membrane proteins. The interaction and co-localization between eNOS and Cav-1 were determined using co-immunoprec-ipitates and immunofluorescence analysis, respectively. Results Different concentrations filipin did not influence the expression of eNOS and p-eNOS protein in HUVECs. In the presence of Ca^2＋, the CaR agonist spermine at concentration of 2.0 mmol/L resulted in an increase in the p-eNOS in HUVECs （P<0.05）, the effect of spermine on the increase of p-eNOS was also completely blocked after acute caveolae disruption with filipin （1.5 mg/L） or transfected with Cav-1 ShRNA （P<0.05）. The expression of the eNOS membrane protein was decreased in HUVECs after cells were treated by filipin （1.5 mg/L） or transfected with Cav-1 ShRNA. Simultaneously, total protein level of eNOS was unaffected. Immunocytochemical results demonstrated that filipin （1.5 mg/L） or transfected with Cav-1 ShRNA decreased eNOS localization in caveolae, increased in the local area surrounding the nucleus. Compared with control group and spermine＋Ca^2＋ group, the interaction of eNOS and Cav-1 in Cav-1 ShRNA group was attenuated （P<0.05）. Conclusions Cav-1 might promote CaR-induced eNOS activation. The mechanisms are involved in the effect of Cav-1 on eNOS localization at the plasma membrane and the inhibition of eNOS translocation to the organelles.