The effects of cellular prion protein on rapid eye movement sleep deprivation-induced spatial memory impairment were investigated, and the related mechanisms explored. Male C57BL/6 mice were randomly divided into four groups: environment control, sleep deprivation control, sleep-deprived-plasmid adeno-associated virus-green fluorescent protein group, and sleep-deprived-plasmid adeno-associated virus-cellular prion protein-green fluorescent protein group. Overexpression of cellular prion protein was induced by stereotaxic injection of adeno-associated viral plasmids-CAG-enhanced green fluorescent protein-cellular prion protein-Flag (a small label, which can be detected with corresponding tagged antibodies) into the hippocampus. Sleep-deprived mice were allowed no rapid eye movement sleep for 72 hours. Morris water maze was used to assess the effects of cellular prion protein on spatial learning and memory. The expression of amyloid-β was also investigated in all groups. The sleep-deprived- plasmid adeno-associated virus- cellular prion protein-green fluorescent protein group spent significantly more time in a goal quadrant compared with the sleep-deprived- plasmid adeno-associated virus-green fluorescent protein group. Sleep deprivation resulted in increased amyloid-β in the hippocampus, which was reversed by the overexpression of hippocampus cellular prion protein. Overexpression of cellular prion protein in the hippocampus rescues rapid eye movement sleep deprivation-induced spatial memory impairment in mice. It is shown that amyloid-β in the hippocampus might be one of the mechanisms.