Prenatal exposure to a high-fat diet (HFD) or microplastics can impact liver fat accumulation in offspring. This study investigates the protective effects of prenatal melatonin on liver injury in male pups resulting from maternal exposure to a HFD and microplastics. Pregnant Sprague-Dawley rats were fed either an HFD or a normal chow diet, with some groups exposed to microplastics alone or in combination with melatonin. Male pups were evaluated on postnatal day 7. Results indicated that pups in the HFD-microplastics group (HFD-Mi) exhibited increased liver lipid accumulation (observed in histological staining), apoptosis (elevated cleaved caspase 3, phospho-AKT, and TUNEL staining), inflammation (higher IL- 6 and TNF-α), and oxidative stress (elevated malondialdehyde). Conversely, melatonin treatment (HFD-Mi + M) significantly reduced these effects, including lipid accumulation, apoptosis, and inflammation, while enhancing antioxidant enzyme glutathione peroxidase activity and improving lipid metabolism (reduced SREBP- 1 expression). These findings suggest that prenatal melatonin mitigates liver injury caused by maternal HFD and microplastics through its anti-inflammatory, antioxidative, and lipid-regulating properties, underscoring its potential hepatoprotective role.