Caveolin-1 (Cav-1) regulates agonist-induced Ca²⁺ entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (N-terminal residues 82 - 101; CSD) interacts with TRPC1 and IP₃R3 to regulate Ca²⁺ entry. We have shown previously the TRPC1 C-terminal residues 781-789 binds to CSD. In this study, we show the TRPC1 C-terminal residues 781-789 truncated (TRPC1-C781-789) mutant expression abolished Ca²⁺ store release-induced Ca²⁺ influx in human dermal endothelial cell line (HMEC) and HEK293 cells. To understand the basis of loss Ca²⁺ influx, we determined TRPC1 binding to IP₃R3. We observed the WT-TRPC1 but not TRPC1-C781-789 effectively interacted with IP3R3. Similarly, WT-TRPC1 interacted with Cav-1 whereas TRPC1-C781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed the WT-Cav-1 but not the Cav-1CSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1CSD was reduced, we measured Ca²⁺ store release-induced Ca²⁺ influx in Cav-1CSD transfected cells. Surprisingly, Cav-1CSD expression showed a gain-of-function in Ca²⁺ entry in HMEC and HEK293 cells. We observed a similar gain-of-function in Ca²⁺ entry when Cav-1CSD was expressed in Cav-1 knock out mice lung endothelial cells. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1CSD interacted with IP₃R3. Further, we observed using confocal imaging the co-localization of IP₃R3 with WT-Cav-1 but not with Cav-1CSD upon Ca²⁺store release in endothelial cells. These findings suggest Cav-1 scaffold domain interacts with TRPC1 and IP₃R3 and thereby regulates Ca²⁺ store release-induced Ca²⁺ entry in endothelial cells.