Objective To investigate the effects of acute sleep deprivation on the behavior and synaptic protein expression of rats. Methods Seventy healthy male Wistar rats were randomly divided into seven groups, a Control group and sleep deprivation groups (24, 48, 72, 96, 120 and 144 hours). The sleep deprivation rat model was established by the modified multiplatform water environment sleep deprivation method. Spatial learning and memory were assessed by the Morris water maze. Anxiety was assessed by the open field test. The morphology and quantity of hippocampal neurons were observed by Nissl staining. Western blot and Real-time PCR were used to determine the expression of synaptophysin (SYN), post-synaptic density protein-95 ( PSD-95), and brain-derived neurotrophic factor ( BDNF) in rats. Results Compared with the Control group, the numbers of standing and modification were significantly increased by prolongation of the sleep deprivation time (P<0. 05). The escape latency and path length were significantly increased in 120 and 144 h groups ( P<0. 05), whereas the number of platform crossings and the percentage of the target quadrant time were significantly decreased (P<0. 01) and negatively correlated to the sleep deprivation time. The expression levels of BDNF,SYN, and PSD-95 were significantly decreased with the prolongation of sleep deprivation time (P<0. 01). Conclusions With the increase in sleep deprivation time, cognitive dysfunction and anxiety gradually deteriorated, which may be related to decreases in the expression of synaptic biomarkers.