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The cell signaling molecules nitric oxide (NO) and Ca2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multicomponent protein assemblies behave within the signaling complexes upon the interplay between NO and Ca2+ signals. Here we demonstrate that TRPC5 channels activated by the stimulation of G-protein–coupled ATP receptors mediate Ca2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1–binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca2+ influx and calmodulin. This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and calmodulin determines the ensemble of Ca2+ mobilization and NO production in vascular endothelial cells. [ABSTRACT FROM AUTHOR]