Astrocytes regulate neuronal excitability by maintaining ion homeostasis and secreting neuroactive substances. We previously showed that blockade or expressional knockdown of inwardly rectifying potassium (Kir) 4.1 channels elevate BDNF expression in astrocytes (Int. J. Mol. Sci., 19, 3313, 2018). In order to explore the neural factors influencing on the Kir4.1-BDNF system, we investigated the effects of acetylcholinergic agents on mRNA expression of Kir4.1 and BDNF in primary cultured astrocytes. Treatment of astrocytes with acetylcholine inhibited Kir4.1 expression and increased BDNF expression in a concentration-related manner. Both inhibition of Kir4.1 and enhancement of BDNF expression by acetylcholine were antagonized by mACh antagonist atropine, but not by nACh antagonist mecamylamine. In addition, inhibition of Kir4.1 expression by acetylcholine was significantly antagonized by the selective M₁ antagonist pirenzepine, which also inhibited acetylcholine-induced BDNF expression. The present results strongly suggest that acetylcholine inhibits Kir4.1 expression and increases BDNF expression via the activation of M₁ receptor in astrocytes.