Aim To study the function of TRPC1 and Orai1 in store and receptor-operated Ca²⁺ entry and nitric oxide generation by SOC and ROC in human umbilical vein endothelial cell. Methods HUVEC were collected and cultured to the second-third passage. We silenced the expression of their genes in HUVEC by transfection constructed TRPC1 or Orai1 RNA interference plasmids. The interference efficiency of their proteins and mRNA levels were determined by Western blot and real-time PCR, respectively. The cell were incubated with CaR agonist spermine, CaR negative allosteric modulator Calhex231 and ROC analogue TPA, protein kinase C (PKC) inhibitor Ro31-8220, PKCs and PKCμ inhibitor Go6967. Intracellular Ca²⁺ concentration ( [Ca²⁺]i) was detected using the fluorescence Ca²⁺ indicator Fura-2/AM, the production of NO was determined by DAF-FM of every group in HUVEC. Results Compared with control group, shRNA targeted to the TRPC1 or Orai1 genes decreased their mRNA and protein levels, respectively (P<0.05)； The results of their mRNA levels by 84.50% and 76.10% and proteins levels were decreased by 83.98% and 71.73%； In four different treatment under the action of factors, the [Ca²⁺]_i and the net NO fluorescence intensity ratio values of transfection of TRPC1shRNA and Orai1shRNA group were significantly reduced (P<0.05). Conclusion TRPC1 and Orai1 participated in CaR-mediated Ca²⁺ influx and NO production activation mediated by SOC and ROC in HUVEC.