Objective: To investigate the effects of acute sleep deprivation on the behavior and synaptic protein expression in rats at different times. Methods: Seventy healthy male Wistar rats were randomly divided into 7 groups, control group, different time sleep deprivation groups (24 hour, 48 hour, 72 hour, 96 hour, 120 hour and 144 hour). The sleep deprivation rat model was established by modified multi-platform water environment sleep deprivation method. Spatial learning and memory was detected by Morris water maze, anxiety was detected by open field test, and the morphology and quantity of hippocampal neurons were observed by Nissl staining. Western blotting and Real-time PCR determined the expressions 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 all significantly increased accompanied by the prolongation of sleep deprivation time (P<0.05). The escape latency and path length significantly increased in rats in the 120 h and 144 h groups (P<0.05), while the number of crossing platforms and the percentage of target quadrant time all significantly decreased (P<0.01), and were negatively correlated with sleep deprivation time. The expression levels of BDNF, SYN and PSD-95 all significantly decreased accompanied by the prolongation of sleep deprivation time (P<0.01), and was negatively correlated with sleep deprivation time. Conclusion: With the increase of sleep deprivation time, cognitive dysfunction and anxiety gradually deteriorated, which may be related to decrease in expression level of synaptic biomarkers.