Aim Serotonergic neurons in the dorsal raphe nucleus (DRN) are involved in the control of sleepwake states.Our previous study indicated that calcium (Ca2+) modulation in the DRN plays an important role in rapid eye movement sleep (REMS) and nonREMS (NREMS) regulation during pentobarbital hypnosis.Present study was designed to investigate the effects of Ca2+ in the DRN on sleepwake regulation and related neuronal mechanism in freely moving rats.Methods CaCl2 (25 or 50 nmol) was microinjected into the DRN and sleep parameters were detective by electroencephalogram (EEG) Monoamine neurotransmitters were detected by HPLCECD.cFos expression in sleepwake regulating nucleus was detected by immunohistochemistry.Results Our results showed that microinjection of CaCl2 (25 or 50 nmol) into the DRN promoted wakefulness and suppressed NREMS including slow wave sleep (SWS) and REMS in freely moving rats.Meanwhile introDRN application of CaCl2 (25 or 50 nmol) significantly increased serotonin in DRN and hypothalamus, as well increased noradrenaline in locus coeruleus (LC) and hypothalamus.Immunohistochemistry study indicated that the application CaCl2 (25 or50 nmol) into the DRN significantly increased cFos expression ratio in wakepromoting neurons including serotonergic neurons in the DRN, noradrenergic neurons in the LC and orxinergic neurons in the perifornical nucleus(Pef), but decreased cFos expression ratio of GABAergic sleeppromoting neurons in the ventrolateral preoptic nucleus (VLPO).Conclusion These results suggest that Ca2+ in the DRN exert arousal effects via upregulation of serotonergic function in endogenous sleepwake regulating pathways.