The regulation of cytosolic free Ca2+ is crucial for controlling smooth muscle contraction. Ca2+ release from sarcoplasmic reticulum (SR) stores contribute to the activation of Ca2+ sensitive ion channels located proximally in the plasma membrane. Regulation of these channels governs membrane potential and the opening of voltage‐gated Ca2+ channels, ultimately leading to muscle contraction. The stromal‐interacting molecule (STIM) 1 acts as the Ca2+‐sensing protein, and is important for the refilling of SR Ca2+ stores. In the current study, we use RNAi technology to further characterize the role of STIM1 in caffeine‐induced SR Ca2+‐depletion and arterial constriction. Treatment of intact cerebral arteries with STIM1 siRNA decreased mRNA expression by ~70% compared with controls. Using ratiometric Ca2+ imaging, we found that caffeine‐induced increases in cytosolic Ca2+ levels were attenuated in STIM1 siRNA‐treated vessels. Furthermore, using simultaneous video dimensional analysis we found that transient caffeine‐induced constriction of arteries treated with STIM1 siRNA was blunted compared with controls. These data suggest that STIM1 plays a central role in regulating SR Ca2+ stores responsible for smooth muscle contraction and the maintenance of arterial tone. RO1HL091905 (SE); F31HL094145‐01 (AG)