Key points The muscarinic acetylcholine receptor (mAChR)‐mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)‐related acid‐sensitive K+ (TASK)1 channels. In this study we focused on the molecular mechanism of mAChR‐mediated inhibition of TASK1 channels. Exposure to muscarine resulted in a clathrin‐dependent endocytosis of TASK1 channels following activation of the muscarinic M1 receptor (M1R). This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non‐receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1. The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin‐dependent manner in response to M1R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells. Abstract Activation of muscarinic receptor (mAChR) in rat adrenal medullary (AM) cells induces depolarization through the inhibition of TWIK‐related acid‐sensitive K+ (TASK)1 channels. Here, pharmacological and immunological approaches were used to elucidate the molecular mechanism for this mAChR‐mediated inhibition. TASK1‐like immunoreactive (IR) material was mainly located at the cell periphery in dissociated rat AM cells, and its majority was internalized in response to muscarine. The muscarine‐induced inward current and translocation of TASK1 were suppressed by dynasore, a dynamin inhibitor. The muscarinic translocation was suppressed by MT7, a specific M1 antagonist, and the dose–response curves for muscarinic agonist‐induced translocation were similar to those for the muscarinic inhibition of TASK1 currents. The muscarine‐induced inward current and/or translocation of TASK1 were suppressed by inhibitors for phospholipase C (PLC), protein kinase C (PKC), and/or Src. TASK1 channels in AM cells and PC12 cells were transiently associated with Src and were tyrosine phosphorylated in response to muscarinic stimulation. After internalization, TASK1 channels were quickly dephosphorylated even while they remained in the cytoplasm. The cytoplasmic TASK1‐like IR material quickly recycled back to the cell periphery after muscarine stimulation for 0.5 min, but not 10 min. We conclude that M1R stimulation results in internalization of TASK1 channels through the PLC–PKC–Src pathway with the consequent phosphorylation of tyrosine and that this M1R‐mediated internalization is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells.