Store-operated Ca²⁺ entry channels (SOCs) play an important role in the regulation of diverse non-excitable cell functions. However, the precise mechanism of SOCs activation is still controversial. Uridine 5'-triphosphate (UTP) was shown to induce Ca²⁺ entry in a dihydropyridines-insensitive manner and accelerated steroidogenesis in bovine adrenocortical fasciculata cells (BAFCs) via the Gq/11 protein-coupled P2Y₂ receptor. Therefore we investigated whether UTP is involved in SOCs activation and the mechanism of UTP-induced SOCs activation. Fura 2-loaded BAFCs were used for the measurement of intracellular concentration of Ca²⁺ ([Ca²⁺]_i) mobilization. Extracellular UTP evoked Ca²⁺ release from intracellular stores followed by an increase in Ca²⁺ entry. The Ca²⁺ influx elicited by UTP was inhibited not by nifedipine, but by Zn²⁺, Cd²⁺, and Ni²⁺ (potency order: Zn²⁺ > Cd²⁺ >> Ni²⁺), and the effect of UTP was also attenuated by a phospholipase C inhibitor (U73122). These results indicate that UTP activates SOCs in BAFCs. The increase in [Ca²⁺]_i by UTP was attenuated by ML-9, a myosin-light chain kinase inhibitor, and calmodulin inhibitors, W-7 and E6 berbamine, in a concentration-dependent manner. These reagents depolymerized actin filaments with rhodamine staining in BAFCs. Cytochalasin D also inhibited UTP-activated SOCs and depolymerized actin filaments. From these results, we proposed that calcium/calmodulin dependent myosin-light chain kinase is involved in the mobilization of actin filaments and the integrity of actin-network plays an important role in UTP-induced SOCs activation in BAFCs.