Introduction: Valproic acid (VPA), a commonly used antiepileptic drug, can induce testicular oxidative stress and injury. Altered autophagic response usually follows testicular injury. Aim of the work: The study aims to evaluate the role of autophagy in the protective effect of the antioxidant Vitamin E (Vit E) against VPA-induced testicular injury. Materials and methods: VPA (100, 300, and 500 mg/kg/day) was administered for 8 days. The protective group received both Vit E (50 mg/kg) and VPA (500 mg/kg). The testicular weight, sperm analysis, and serum testosterone concentration, as well as testicular histopathology, steroidogenic gene expression, and oxidative stress markers were evaluated. The mRNA or protein expression of autophagy-related proteins [adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), microtubule associated protein light chain 3 (LC3), Beclin1, and p62] were measured using RT-PCR or immunohistochemistry. Results: VPA resulted in lower testes weight and sperm quality with aberrant morphology. VPA dose dependently induced testicular oxidative stress, which was associated with decreased steroidogenic gene expression and serum testosterone levels, as well as deteriorated histopathology. These biochemical and histological changes were also associated with autophagy induction (higher LC3 and Beclin1 and lower p62) that was lost with the highest toxic dose (500 mg/kg). The attenuated autophagy with the highest dose was accompanied by AMPK downregulation and mTOR upregulation. Vit E protected against VPA-mediated oxidative stress and toxicity. while also restoring autophagic response and AMPK/mTOR levels. Conclusions: The study highlights vitamin E as a valuable protective asset against VPA-induced testicular injury, possibly through AMPK-mTOR-dependent autophagy induction.