Stromal interaction molecule (STIM1) and its isoform (STIM2) are single span sarco/endoplasmic (SR) transmembrane proteins that function as powerful SR Ca2+ sensors. When the SR Ca2+ content decreases STIM proteins migrate in proximity of the plasma membrane to tether and activate the Orai channels initiating the so called store operated Ca2+ entry (SOCE). In non-excitable cells STIM mediates Ca2+ entry that is required for regulating cell proliferation and migration. Smooth muscle cells (SMC) can exist as non-excitable cells, known also as the “proliferative” phenotype, or as excitable cells, known as the “contractile” phenotype. Furthermore, SMC can interchange their phenotype in response to environmental stimuli; Ca2+ signaling plays a crucial role in regulating this transition. However, very little is known about the role of STIM in SMC. Because isolated primary SMC quickly lose their contractile phenotype when placed in culture, the role of STIM proteins in SMC has been eluded. To overcome this limitation we used the Cre-lox technology approach to generate SM-specific STIM1-, STIM2-, and STIM1/STIM2- knockout (KO) mice, this model allowed us to systematically analyze the physiological role of STIM in SMC. SM-STIM1-KO mice survival rate was only about 50% within the first 30 days after birth. In addition, SM-STIM1-KO mice showed a consistent reduced body weight when compared to control mice. While the SM-STIM1/STIM2 double-KO phenotype was perinatally lethal, the SM-STIM2-KO was without a detectable phenotype. However, in the SM-STIM1 KO mice the STIM2 expression is enough to rescue the otherwise lethal phenotype, revealing that also STIM2 plays an important role in the SMC. Smooth muscle containing organs, such as intestine and aorta harvested from SM-STIM1-KO mice revealed morphological abnormalities when compared with organs harvested from control mice. Vascular reactivity analyzed using wire myography revealed that while depolarization-induced aortic contraction was unchanged, phenylephrine-mediated contraction was reduced by 26%, and store-dependent contraction was almost eliminated in aortas isolated from SM-STIM1-KO mice. Neointima formation induced by partial carotid artery ligation was suppressed by 54%. Consistently, in vitro PDGF-induced SMC proliferation was also reduced by 79% in STIM1-KO SMC. Notably, the Ca2+ store-refilling rate in STIM1-KO SMCs was substantially reduced, and sustained PDGF-induced Ca2+ entry was abolished. This defective Ca2+ homeostasis prevents PDGF-induced NFAT activation in both contractile and proliferating SMCs. In conclusion, our data show that STIM1-regulated Ca2+ homeostasis is crucial for NFAT-mediated transcriptional control required for induction of SMC proliferation, development, and growth during physiological as well as pathophysiological conditions.