<p>Chronic stress is a major contributor to male reproductive dysfunction leading to testicular damage and impaired spermatogenesis. This study investigates the protective effects of β-sitosterol, a phytosterol with known antioxidant properties, against CUS-induced testicular damage in rats. Male Wistar rats were divided into Control, Chronic Unpredictable Stress (CUS), and CUS + β-sitosterol. The CUS and CUS + β-sitosterol groups were exposed to random stressors for eight weeks. β-Sitosterol was administered orally at a dose of 20&#xa0;mg/kg for three weeks, starting from the fifth week of CUS induction. Behavioral tests like EPMT and NSFT were conducted to confirm CUS induction, after which serum, testis, and epididymis samples were collected for analysis. β-sitosterol significantly increased testis and epididymis weight, along with sperm counts in CUS rats. Histological analysis revealed restoration of testicular cellular structure, as indicated by an improved Johnsen's index scores. Additionally, β-sitosterol restored antioxidant levels and oxidative stress parameters in testicular tissue. TEM showed germ cell integrity and restored basement membrane structure in the CUS + β-sitosterol group. In silico analysis indicated strong interactions of β-sitosterol with FNDC5, P450scc, and 3β-HSD proteins involved in steroidogenesis. Immunohistochemistry confirmed an increased expression of FNDC5 in the CUS + β-sitosterol group, β-sitosterol ameliorates CUS-induced testicular damage and improves sperm count, highlighting its potential as a dietary intervention for stress-related male infertility. Further preclinical and clinical studies are warranted to explore its therapeutic potential.</p> Graphical Abstract <p></p>