The non-neuronal cholinergic system, widely distributed in nature, is an ancient system that has not been well studied in insects. This study aims to investigate the key components of the cholinergic system and to identify the non-neuronal acetylcholine (ACh)-producing cells and the acting sites of ACh in the Malpighian tubules (MTs) of Mythimna separata. We found that non-neuronal ACh in MTs is synthesized by carnitine acetyltransferase (CarAT), rather than choline acetyltransferase (ChAT), as confirmed by using enzyme inhibitors and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Fluorescence in situ hybridization revealed the presence of CarAT mRNA within MTs, specifically localized in the principal cells. Immunohistochemistry showed strong staining for A-mAChR, a muscarinic acetylcholine receptor, in the principal cells. Pharmacological analysis further demonstrated that ACh acts through A-mAChR in the principal cells to increase the intracellular Ca²⁺ concentration. These findings provide compelling evidence for the existence of a non-neuronal cholinergic system in the MTs of M. separata, and the principal cells play a crucial role in ACh synthesis via CarAT.