The tight regulation of endothelial barrier function is of prime importance for physiology and, when this barrier is compromised, it contributes to pathophysiology. Endothelial cells that line the vasculature change shape, and monolayers of these cells react to thrombin with increased permeability. In vivo, this increased permeability allows for transudation of plasma proteins and edema formation. The endoplasmic reticulum-resident calcium (Ca2+) sensor stromal interacton molecule 1 (STIM1) is intimately involved in the cellular response to thrombin. In human endothelial cells, STIM1 fulfills its role within the thrombin signaling cascade independently of store-operated Ca2+ entry (SOCE) through Orai channels, and thus STIM1 emerges as a signaling component linking the thrombin receptor to RhoA activation and stress fiber formation. This SOCE-independent role of STIM1 highlights a surprising new aspect of this multifaceted cellular regulator.