<p>Hispidulin, a naturally occurring flavonoid found in various medicinal plants, exhibits anti-proliferative effects in multiple cancer models; however, its role in regulating intracellular calcium (Ca²⁺) signaling in human breast cancer cells remains unclear. Here, we investigated its effects on intracellular Ca²⁺ dynamics, cytotoxicity, and Ca²⁺-associated signaling in T-47D human breast cancer cells. Hispidulin (40–120&#xa0;μM) induced a concentration-dependent increase in intracellular Ca²⁺ levels ([Ca²⁺]_i) accompanied by reduced cell viability, and pretreatment with the intracellular Ca²⁺ chelator BAPTA-AM further enhanced cytotoxicity, indicating that disruption of Ca²⁺ homeostasis potentiates cell death. Removal of extracellular Ca²⁺ partially attenuated the response, with additional inhibition by nifedipine, a dihydropyridine-sensitive Ca²⁺ channel blocker, suggesting involvement of voltage-dependent Ca²⁺ influx. The Ca²⁺ response was also reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of store-operated Ca²⁺ entry (SOCE), and by the protein kinase C (PKC) inhibitor GF109203X, implicating SOCE and PKC signaling. Thapsigargin-induced depletion of endoplasmic reticulum (ER) Ca²⁺ stores indicated that hispidulin mobilizes ER Ca²⁺, while phospholipase C (PLC) inhibition by U73122 completely abolished the [Ca²⁺]_i increase. Collectively, these findings demonstrate that hispidulin regulates intracellular Ca²⁺ homeostasis via PLC-dependent ER Ca²⁺ release and extracellular Ca²⁺ influx through SOCE and nifedipine-sensitive Ca²⁺ entry components, leading to PKC activation. Enhanced cytotoxicity following Ca²⁺ chelation further underscores the importance of Ca²⁺ homeostasis in determining cellular responses to hispidulin.</p>