Environmental nanoplastics (NPs) have harmful effects on health. This study investigated the effects of polystyrene (PS) NPs on steatosis and fatty liver disease. PS-NP oral administration, in conjunction with a high-fat diet (HFD), synergistically exacerbated the symptoms of steatosis in mice, leading to increased alanine transaminase, aspartate aminotransferase, and cholesterol levels; no effects were observed with PS-NPs on a normal chow diet. Transcriptome analysis unveiled that PS-NPs interfered with actin organization, cell-cell adhesion, PPAR signaling, and lipid metabolism. In HepaRG cells, PS-NPs rapidly entered by inducing actin rearrangement, resulting in the formation of numerous small cytoplasmic vesicles. This treatment led to an augmented number of acidic organelles, leading to development and buildup of large vacuoles, indicative of enlarged pre-lysosomal and lysosomal compartments. PS-NP exposure hampered p62 degradation, leading to LC3B accumulation and decreased cathepsin B and D activity. Additionally, PS-NP exposure resulted in accumulation of lipid droplets and elevated expression of lipogenesis-, transport-, and storage-related genes. These findings suggest that excessive endocytosis driven by PS-NPs worsens MASLD in HFD through accumulation of lysosomes and large vacuoles with reduced cathepsin activity.