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The habenular nucleus (Hb) is an important structure that regulates the function of the pineal gland,which may affect melatonin content in the pineal gland after sleep deprivation (SD). In the present study, high performance liquid chromatography showed that the melatonin content in the pineal gland was significantly reduced, and Y-aminobutyric acid content in the Hb was significantly increased after SD. Furthermore, the melatonin content in the pineal gland was markedly reduced after Hb lesion under normal sleep and SD conditions. Immunohistochemistry showed that the number of Fos-positive neurons was significantly decreased in the lateral and medial Hb after SD.The findings demonstrate that the reduction of melatonin in the pineal gland after SD is related to decreased activity of Hb neurons, and that the Hb can regulate sleep-wake rhythm by influencing melatonin secretion in the pineal gland.