AbstractCinobufagin, a bufadienolide of toad venom of Bufo bufo gargarizans, is used as a cardiotonic, central nervous system (CNS) respiratory agent, as well as an analgesic and anesthetic. However, several research showed that bufadienolide has a few side effects on the CNS, such as breathlessness or coma. Although cinobufagin was shown to display pharmacological effects in various models, the toxic effect of cinobufagin is elusive in brain cell models. The aim of this study was to explore whether cinobufagin affected viability, Ca2+ homeostasis, and reactive oxygen species (ROS) production in Gibco® Human Astrocyte (GHA) and HCN‐2 neuronal cell line. In GHA cells but not in HCN‐2 cells, cinobufagin (20–60 μM) induced [Ca2+]i rises. In terms of cell viability, chelation of cytosolic Ca2+ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N'N'‐tetraacetic acid reduced cinobufagin‐induced cytotoxicity in GHA cells. In GHA cells, cinobufagin‐induced Ca2+ entry was inhibited by 2‐aminoethoxydiphenyl borate or SKF96365. In a Ca2+‐free medium, treatment with thapsigargin or U73122 abolished cinobufagin‐evoked [Ca2+]i rises. Furthermore, treatment with N‐acetylcysteine reversed ROS production and cytotoxicity in cinobufagin‐treated GHA cells. Together, in GHA cells but not in HCN‐2 cells cinobufagin caused cytotoxicity that was linked to preceding [Ca2+]i rises by Ca2+ influx via store‐operated Ca2+ entry and phospholipase C‐dependent Ca2+ release from the endoplasmic reticulum. Moreover, cinobufagin induced ROS‐associated cytotoxicity.