Apoptosis plays a critical role in the maintenance of gut mucosal epithelial homeostasis and is tightly regulated by numerous factors including intracellular Ca²⁺. Transient receptor potential canonical-1 (TRPC1) is expressed in intestinal epithelial cells (IECs) and functions as a store-operated Ca²⁺ channel. We have recently demonstrated that increased TRPC1 activity sensitizes IECs to apoptosis, but the upstream signaling initiating TRPC1 activation remains elusive. The novel protein, STIM1, is shown to act as a store Ca²⁺ sensor and it can rapidly translocate to the plasma membrane (PM) where it directly interacts with TRPC1. The current study determined whether STIM1 plays an important role in the regulation of IEC apoptosis by activating TRPC1 channel activity. Methods: Studies were conducted in IEC-6 cells (derived from rat intestinal crypts) and stable TRPC1-transfected IECs (IEC-TRPC1). Apoptosis was induced by tumor necrosis factor-α (TNFα)/cycloheximide (CHX), and intracellular free Ca²⁺ concentration ([Ca²⁺]_cyt) was measured by fluorescence digital imaging analysis. Functions of STIM1 were investigated by specific siRNA (siSTIM1) and ectopic overexpression of the constitutively active STIM1 EF-hand mutants. Results: Stable STIM1-transfected IEC-6 cells (IEC-STIM1) highly expressed STIM1 protein (~5-folds) and displayed a sustained increase in Ca²⁺ influx after Ca²⁺ store depletion (~2-folds). Susceptibility of IEC-STIM1 cells to TNFα/CHX-induced apoptosis increased significantly as measured by changes in morphological features, DNA fragmentation, and caspase-3 activity. Apoptotic cells were increased from ~30% in parental IEC-6 cells to ~70% in stable IEC-STIM1 cells 4 h after exposure to TNFα/CHX. In addition, stable IEC-TRPC1 cells also exhibited an increased sensitivity to TNFα/CHX-induced apoptosis, which was prevented by STIM1 silencing through siSTIM1 transfection. STIM1 silencing by siSTIM1 also decreased Ca²⁺ influx after store depletion in cells overexpressing TRPC1. Levels of Ca²⁺ influx due to store depletion were decreased by ~70% in STIM1-silenced populations. Similarly, exposure of IEC-STIM1 cells to the Ca²⁺ free medium also blocked increased sensitivity to apoptosis. Conclusions: These results indicate that 1) STIM1 plays an important role in the regulation of IEC apoptosis by altering TRPC1 activity and 2) ectopic STIM1 expression sensitizes IECs to apoptosis through induction in TRPC1-mediated Ca²⁺ influx.