The Ca 2+ ‐sensing stromal interaction molecule (STIM) proteins are crucial Ca 2+ signal coordinators. Cre‐lox technology was used to generate smooth muscle (sm)‐targeted STIM1‐, STIM2‐, and double STIM1/STIM2‐knockout (KO) mouse models, which reveal the essential role of STIM proteins in Ca 2+ homeostasis and their crucial role in controlling function, growth, and development of smooth muscle cells (SMCs). Compared to Cre +/– littermates, sm‐STIM1‐KO mice showed high mortality (50% by 30 d) and reduced bodyweight. While sm‐STIM2‐KO was without detectable phenotype, the STIM1/STIM double‐KO was perinatally lethal, revealing an essential role of STIM1 partially rescued by STIM2. Vascular and intestinal smooth muscle tissues from sm‐STIM1‐KO mice developed abnormally with distended, thinned morphology. While depolarization‐induced aortic contraction was unchanged in sm‐STIM1‐KO mice, α 1 ‐adrenergic‐mediated contraction was 26% reduced, and store‐dependent contraction almost eliminated. Neointimal formation induced by carotid artery ligation was suppressed by 54%, and in vitro PDGF‐induced proliferation was greatly reduced (79%) in sm‐STIM1‐KO. Notably, the Ca 2+ store‐refilling rate in STIM1‐KO SMCs was substantially reduced, and sustained PDGF‐induced Ca 2+ entry was abolished. This defective Ca 2+ homeostasis prevents PDGF‐induced NFAT activation in both contractile and proliferating SMCs. We conclude that STIM1‐regulated Ca 2+ homeostasis is crucial for NFAT‐mediated transcriptional control required for induction of SMC proliferation, development, and growth responses to injury.—Mancarella, S., Potireddy, S., Wang, Y., Gao, H., Gandhirajan, K., Autieri, M., Scalia, R., Cheng, Z., Wang, H., Madesh, M., Houser, S. R., Gill, D. L. Targeted STIM deletion impairs calcium homeostasis, NFAT activation, and growth of smooth muscle. FASEB J. 27, 893–906 (2013). www.fasebj.org