Objective To observe the changes of the neuropeptide S (NPS) expression in dorsomedial nucleus of rat hypothalamus after rapid eye movement (REM) sleep deprivation. Methods Totally 24 adult male rats were evenly randomized into 3 groups: normal control group (CC), tank control group (TC) and REM sleep deprivation group (SD). Modified multiple platform method (MMPM) was used to establish REM sleep deprivation model in rats. Immunohistochemistry and hybridization in situ were applied to examine the expression of NPS protein and mRNA in the rat hypothalamus. Results There was no significant differences in the expression of NPS protein or mRNA in rats hypothalamus between CC group and TC group. After REM sleep deprivation, NPS protein and mRNA positive cells in rats hypothalamus in SD group were (27.86±2.47) and (25.75±2.12), respectively, which were significantly more than those in CC group and TC group (16.75±2.12 and 19.63±1.85 in CC group, 18.60±1.60 and 18.50±1.69 in TC group; P<0.05). Conclusion The expression of NPS is increased in the awakening region of rat hypothalamus after REM sleep deviation, suggesting that NPS might be related to the sleep awakening mechanism.