Sleep is essential for proper cognitive functioning. Ozone can delay both the aging process as well as the associated neurodegeneration. An untested hypothesis is that ozone may play a role in reducing the cognitive impairment associated with sleep deprivation. This study was designed to elucidate the mechanism of ozone’s effect on chronic REM sleep deprivation induced cognitive dysfunction in mice. Ozone ameliorated cognitive dysfunction in chronic REM sleep deprived mice, increased the number of dendritic spines in the hippocampus region and decreased neuronal loss. Administration of ozone may protect against chronic REM sleep deprivation induced cognitive dysfunction by enhancing the expression of Semaphorin 3A (Sema3A) and PlexinA1 concentrations as well as P-CRMP2/CRMP2 in the hippocampus. Moreover, ozone was associated with acetylation of α-tubulin, which, in turn, is associated with microtubule network dynamics and modulates ATP synthase activity. In conclusion, ozone may play a neuroprotective role and improve chronic REM sleep deprivation induced spatial recognition and learning memory dysfunction in mice.