Sho-ju-sen, a Japanese herbal medicine composed of three herbs; kumazasa leaf, Japanese red pine leaf, and ginseng radix, has been used as a nourishing tonic. Sho-ju-sen is reported to improve many symptoms related to vegetative dystonia and unidentified clinical syndrome such as fatigue and depression. Here we report the effects of Sho-ju-sen and its components on catecholamine secretion in cultured bovine adrenal medullary cells. We found that Sho-ju-sen inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue, in a concentration (0.5-1.0%)-dependent manner. Extract of kumazasa leaves, but not ginseng radixes and red pine leaves, concentration-dependently (0.5-1.0%) inhibited catecholamine secretion induced by acetylcholine. Sho-ju-sen and its component, kumazasa leaves, also attenuated ACh-induced ⁴⁵Ca²⁺ influx. Sho-ju-sen and kumazasa leaves (0.1- or 0.5-1.0%) directly inhibited the Na⁺ current evoked by acetylcholine in Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors. The present findings suggest that Sho-ju-sen and its component, kumazasa leaves, inhibit catecholamine secretion and Ca²⁺ influx via inhibition of Na⁺ influx mediated through nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.