Mesaconitine, one of Aconitum carmichaelii Debx bioactive compounds, was shown to evoke Ca(2+) homeostasis and its related physiological effects in endothelial cell types. However, the effect of mesaconitine on Ca(2+) signaling and cell viability in human brain microvascular endothelial cells is unclear. This study focused on exploring whether mesaconitine changed cytosolic Ca(2+) concentrations ([Ca(2+)](i)), affected cell viability, and established the relationship between Ca(2+) signaling and viability in HBEC-5i human brain microvascular endothelial cells. In HBEC-5i cells, cell viability was measured by the cell proliferation reagent (WST-1). [Ca(2+)](i) was measured by the Ca(2+)-sensitive fluorescent dye fura-2. Mesaconitine (10-100 muM) concentration dependently induced [Ca(2+)](i) rises. Ca(2+) removal reduced the signal by approximately 25%. Mesaconitine (40-100 muM) caused cytotoxicity in HBEC-5i cells. This cytotoxic response was significantly reversed by chelation of cytosolic Ca(2+) with BAPTA/AM. In Ca(2+)-containing medium, mesaconitine-induced Ca(2+) entry was inhibited by 25% by modulators of store-operated Ca(2+) channels and protein kinase C (PKC). Furthermore, mesaconitine also induced Mn(2+) influx suggesting of Ca(2+) entry. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin abolished mesaconitine-evoked [Ca(2+)](i) rises. Conversely, treatment with mesaconitine abolished thapsigargin-evoked [Ca(2+)](i) rises. Inhibition of phospholipase C (PLC) with U73122 abolished mesaconitine-induced [Ca(2+)](i) rises. In sum, mesaconitine caused cytotoxicity that was triggered by preceding [Ca(2+)](i) rises. Furthermore, mesaconitine induced [Ca(2+)](i) rises by evoking Ca(2+) entry via PKC-sensitive store-operated Ca(2+) channels and PLC-dependent Ca(2+) release from the endoplasmic reticulum. It suggests that Ca(2+) signaling have a potential cytotoxic effect on mesaconitine-treated human brain microvascular endothelial cells.