BACKGROUND: The egg-laying performance of hens is primarily regulated by ovarian follicle growth and development; these follicles are susceptible to oxidative damage caused by excessive reactive oxygen species (ROS). Oxidative damage can lead to follicular atresia and impaired reproductive performance. Melatonin (MT), a known endogenous antioxidant, plays a role in regulating oxidative damage, but its precise mechanisms in mitigating HO-induced oxidative damage via mitophagy regulation in granulosa cells remain unclear. METHODS: An in vitro oxidative damage model was established by determining the optimal HO concentration using CCK-8 fluorescence quantification. The optimal MT concentration was identified through fluorescence quantification and catalase (CAT) activity assays. The protective effects of MT against HO-induced oxidative damage in follicular granulosa cells were investigated using flow cytometry, Western blotting, ELISA, and quantitative fluorescence analysis. RESULTS: An in vitro oxidative damage model was established using HO-induced granulosa cells, characterized by and upregulation and and downregulation. The optimal MT concentration for reducing cellular injury was determined. MT co-treatment enhanced CAT, GSH, and SOD activities, decreased LC3-II/LC3-I conversion, and increased P62 expression. Furthermore, MT reduced autophagic vesicle formation and restored mitochondrial membrane potential, demonstrating its protective effect against HO-induced oxidative damage. CONCLUSIONS: Melatonin alleviates HO-induced oxidative damage in chicken follicular granulosa cells by modulating antioxidant defense, autophagy, and mitochondrial function. These findings provide newer insights to our understanding of the regulatory mechanisms underlying the alleviation of the HO-induced oxidative damage in granulosa cells during ovarian follicle development in chickens.