Cyclophosphamide (Cy), a chemotherapeutic agent, is widely used to treat tumoursand is also associated with premature ovarian insufficiency. 4-Hydroperoxycyclophosphamide (4-HC), an active metabolite of Cy, was used for in vitro experiments. Granulosa cells (GCs) are crucial for maintaining follicle development and are also used in reproductive toxicity research in vitro. Resveratrol (Res), a polyphenolic compound, exhibits multiple effects in cells and animal models. To date, whether Res pretreatment has a protective effect on GCs induced by Cy remains unclear. This was an in vitro study, and primary cultures of rat GCs were used. Rat GCs were treated with 4-HC alone, Res + 4-HC or Res + 4-HC + EX527, and GCs survival rates, oxidative stress levels, apoptosis rates and related Sirt1 pathway proteins were evaluated. We demonstrated that 4-HC caused GC damage by increasing oxidative stress, autophagy and apoptosis. Res pretreatment improved 4-HC-induced GC damage by increasing Sirt1 expression, reducing oxidative stress levels and decreasing Beclin1, LC3B, Box and Caspase-3 levels. Importantly, the addition of EX527, which is a selective inhibitor of Sirt1, reversed the protective effect of Res pretreatment, indicating that Sirt1 may be an important mediator of the protective effect of Res. Taken together, we demonstrated that Res may be a potential drug to improve fertility preservation for patients undergoing chemotherapy.