Canonical transient receptor potential 1 (TRPC1) proteins form store-operated Ca2+-permeable ion channels in vascular smooth muscle cells (VSMCs), which are involved in regulating contractility, and cell proliferation and migration (1). However, the mechanism involved in coupling depletion of internal Ca2+ stores to activation of TRPC1 channels remains elusive. The present work investigates the idea that store-depletion triggers Gaq-mediated PLC and TRPC1 channel activities in freshly isolated and primary cultured rabbit and mice mesenteric artery VSMCs, measured using the PIP2 fluorescent sensor PLCδ1-GH-GFP. Application of BAPTA-AM, TPEN or CPA, which all deplete Ca2+ stores, induced translocation of PLCδ1-GH-GFP-mediated signals from the plasma membrane to the cytosol, which were reduced by the PLC inhibitor U73122. BAPTA-AM-mediated translocation of PLCδ1-GH-GFP signals were greatly inhibited by transduction of cells with shRNA STIM1, and were absent in TRPC1-/- VSMCs. Co-immunoprecipitation, immunocytochemistry and proximity ligation assay showed that BAPTA-AM induced translocation of STIM1 from the cytosol to the plasma membrane, where it interacted with TRPC1. Moreover, store-operated STIM1/TRPC1 complexes associated with Gaq and PLCβ1. In contrast, store-operated translocation of PLCδ1-GH-GFP signals, and interactions between STIM1, TRPC1, Gαq and PLCβ1 were not altered in Orai1-/- mice. These findings propose a novel mechanism for store-operated TRPC1 channels in VSMCs, through formation of STIM1/TRPC1 signalling complexes which drive activation of the classical Gq/PLC pathway. Our results argue against a role for Orai1 in the activation of TRPC1 channels in VSMCs.