PURPOSE: Insufficient sleep and insomnia are common issues associated with modern lifestyles that often contribute to the development of mental health disorders. 4-aminopyridine (4-AP), a voltage-gated potassium (Kv) channel antagonist, is commonly used in the treatment of multiple sclerosis (MS). It has been shown to improve nerve conduction velocity, strengthen myelin, and increase axonal area after injury. In addition, 4-AP has been reported to reduce behavioral disorders, including depression. The aim of this study was to investigate the effects of 4-AP on anxiety-like behavior in mice subjected to rapid eye movement (REM) sleep deprivation. METHODS: Fifty male mice were randomly divided into five groups: control, normal saline (NS) (receiving normal saline via gavage), AP-0.25, AP-0.5, and AP-1 (receiving daily doses of 0.25, 0.5, and 1 mg/kg of 4-AP, respectively by gavage). All groups except the control group underwent SD for five consecutive days. The animals' locomotion and anxiety-like behavior were assessed using the open field and elevated plus maze tests. After behavioral testing, N-methyl-D-aspartate receptor (NMDA-R), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R), and tumor necrosis factor (TNF-α) were measured by western blotting, and also malondialdehyde (MDA) and total antioxidant capacity (TAC) were analyzed by ELISA in the hippocampus. FINDING: AP-1 significantly reduced the levels of anxiety-like behavior compared to the NS group in both tests. In AP-1, a significant decrease in the levels of NMDA-R, AMPA-R, TNF-α, and MDA was observed. While these levels were increased in the NS group. In addition, AP-1 showed a higher level of TAC compared to the NS group, indicating an increase in antioxidant levels. CONCLUSION: 4-AP may be effective in reducing anxiety-like behavior in sleep-deprived mice by modifying the levels of NMDA-R, AMPA-R, and TNF-α, while simultaneously reducing oxidative stress induced by sleep deprivation in the hippocampus.