Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in pineal gland. Although substantial amount of Ca²⁺ influx is triggered by NE, the Ca²⁺ entry pathway and its physiological relevance have not been clearly elucidated. Here, we report that the Ca²⁺ influx triggered by NE significantly regulates the protein levels of serotonin N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and was responsible for maintaining the Ca²⁺ responses after repetitive stimulation. The Ca²⁺ entry evoked by NE was dependent on the phospholipase C (PLC) activation. NE evoked substantial amount of Ca²⁺ entry even after cells were treated with oleoyl-acyl-sn-glycerol (OAG), an analog of DAG. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca²⁺ entry. Moreover, NE failed to induce further Ca²⁺ entry after development of Ca²⁺ entry by thapsigargin (TG), suggesting that the pathway of Ca²⁺ entry by NE would be identical to that of TG. Interestingly, Ca²⁺ entry evoked by NE or TG induced membrane hyperpolarization, which was reversed by iberiotoxin (IBTX), a specific inhibitor of large conductance Ca²⁺-activated K⁺ (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, activation of BK channels triggered by NE contributed to regulate the protein level of AANAT. Collectively, these results suggest that NE triggers Ca²⁺ entry coupled to BK channels and that NE-induced Ca²⁺ entry is important for the regulation of AANAT.