OBJECTIVE Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. RESULTS We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. CONCLUSIONS Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.