Aims We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium (Ca2+)‐permeable non‐selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. Methods Western blotting and immunoprecipitation were used for the evaluation of protein phosphorylation; protein phosphatase 2B (PP2B) activity was assessed by convention kit; Griess assay was for NO production; tube formation and Matrigel plug assay were used for angiogenesis. Results In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα) at Serine 657 (Ser657) and PKCβ2 at Ser660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B–PKC complex. Inhibition of TRPV1 activation by the pharmacological antagonists, removal of extracellular Ca2+ or pharmacological inhibition of PI3K/Akt/calmodulin‐dependent protein kinase II/AMP‐activated protein kinase signalling pathway abolished the evodiamine‐induced alterations in phosphorylation of eNOS at Thr497, PKCα at Ser657, PKCβ2 at Ser660 and PP2B activity, as well as the formation of a PP2B–PKC complex. Inhibition of PP2B activation partially reduced the evodiamine‐induced NO bioavailability and tube formation in endothelial cells (ECs) and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα at Ser657 and PKCβ2 at Ser660 in apolipoprotein E (ApoE)‐deficient mouse aortas but not TRPV1‐deficient or ApoE/TRPV1 double‐knockout mice. Conclusion TRPV1 activation in ECs may elicit a Ca2+‐dependent effect on PP2B–PKC signalling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.