Activated protein C (APC) exerts endothelial protein C receptor (EPCR)–dependent neuroprotective effects in a brain focal ischemia model and direct cellular effects on human umbilical vein endothelial cells (HUVECs) via protease-activated receptor-1 (PAR-1). Because PAR receptors are expressed in brain endothelium and mediate intracellular calcium concentration ([Ca²⁺]_i) signaling, we hypothesized that APC may regulate intracellular [Ca²⁺] flux in human brain endothelial cells (BECs) via EPCR and PAR-1. Primary cortical BECs derived from human autopsies (early passage) and HUVECs were used for [Ca²⁺]_i imaging fluorometry. Cells were exposed for 1 minute to APC, protein C zymogen, or mutant Ser360Ala-APC, and [Ca²⁺]_i was monitored in the presence or absence of antibodies against PAR-1, PAR-2, PAR-3, or EPCR. APC, but not protein C zymogen or the active site mutant Ser360Ala-APC, induced dose-dependent [Ca²⁺]_i release in human BECs (Δ[Ca²⁺]_imax = 278.3 ± 19.5 nM; EC₅₀ for APC = 0.23 ± 0.02 nM, n = 70 measurements). APC-induced [Ca²⁺]_i signaling was abolished by a cleavage site blocking anti–PAR-1 antibody, whereas anti–PAR-2 and –PAR-3 antibodies were without effect. Antibody RCR252 that ablates APC binding to EPCR blocked APC-mediated [Ca²⁺]_i signaling, whereas anti-EPCR antibody RCR92 that does not block APC binding did not abolish the APC-induced [Ca²⁺]_i response. Experiments using HUVECs confirmed the findings for BECs. Thapsigargin inhibited the APC-induced [Ca²⁺]_i signal, implicating the endoplasmic reticulum as a major source for the APC-induced [Ca²⁺]_i release. These data suggest that APC regulates [Ca²⁺]_i in human brain endothelium and in HUVECs by binding to EPCR and signaling via PAR-1.