Objective: Sleep deprivation (SD) is a common problem in today’s stressful lifestyle and have physiological consequences, including reproductive dysfunction and infertility. As an antioxidant, olive oil may be effective in reducing testicular and spermatological damage by decreasing the production of free radicals. Methods: This study investigated the effects of olive oil on sperm quality and testicular structure using stereological methods to assess rats with SD. Results: When comparing SD group to grid floor+distilled water (GR) group, we found that the sperm count and motility, as well as the percentage of slow progressive sperm was significantly lower in SD group (p<0.05), but the percentage of immotile sperm was higher (p<0.01). However, no improvement was observed in sperm count or motility after concomitant treatment of SD group with olive oil. Stereological examinations revealed no significant change in the total volumes of the seminiferous tubules, interstitial tissue, and germinal epithelium in the study groups. Conversely, the total number of testicular cell types was significantly lower in SD group than in GR group. Although the total number of Sertoli and Leydig cells was significantly higher in the SD+olive oil group than in the untreated SD group, no significant difference in the total number of other testicular cell types was observed between the two groups. Conclusion SD potentially induced structural changes in testis that affected sperm count and motility. However, olive oil only improved the total number of Sertoli and Leydig cells in the animals with SD and did not improve sperm count and motility.