Microplastics are toxic to aquatic organisms. Metabolic disturbances in the liver are highly correlated with the pathophysiology of diseases and toxicology of contaminants. However, the effects of microplastics on the comprehensive metabolic responses of aquatic animal livers are unclear. Therefore, an untargeted metabolomics approach using gas chromatography–mass spectrometry was employed to discover metabolic disorders in marine medaka (Oryzias melastigmas) liver after microplastic exposure. We found that 83 metabolites were significantly altered in marine medaka liver after 10-μm and/or 200-μm polystyrene microplastic (PS) exposure. Subsequent metabolic pathway analysis revealed that 33 and 28 metabolic pathways, such as glycerolipid metabolism, biosynthesis of unsaturated fatty acids, glycolysis/gluconeogenesis, fructose and mannose metabolism, and glycine, serine and threonine metabolism, were significantly altered after 10-μm (PS-10) and 200-μm (PS-200) PS exposure, respectively. Most monosaccharides (e.g., glucose, mannose, and ribose), organic acids (e.g., lactate, fumarate, and malate) and amino acids (e.g., alanine, serine, and leucine) significantly decreased, while most fatty acids and fatty acid methyl and ethyl esters significantly increased in marine medaka liver due to PS-10 and/or PS-200 exposure. The results indicated that PS exposure triggered inhibition of the monosaccharide metabolism, tricarboxylic acid cycle, glycolysis, pentose phosphate pathway, and amino acid metabolism, and accumulation of fatty acids, fatty acid methyl and ethyl esters in marine medaka liver. The varying influences of PS-10 and PS-200 on hepatic metabolism were observed. Changes in the carbohydrate, amino acid, and lipid metabolism induced by PS-10 were greater than those induced by PS-200. However, PS-200 induced the hepatic accumulation of most disaccharides and trisaccharides, including lactose, maltose, mannobiose and maltotriose, while PS-10 did not. The increased toxicity caused by PS-10 than that by PS-200 may be associated with the hepatic accumulation observed only in the group exposed to PS-10. This study provides novel insights into microplastic ecotoxicology.