The therapeutic effects of melatonin on cholestatic liver injury have received widespread attention recently. The aim of the present study was to investigate the mechanisms of the anti-cholestatic effects of melatonin against α-naphthyl isothiocyanate (ANIT)-induced liver injury in rats and to screen for potential biomarkers of cholestasis through isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Rats orally received melatonin (100 mg/kg body weight) or an equivalent volume of 0.25% carboxymethyl cellulose sodium salt 12 h after intraperitoneal injection of ANIT (75 mg/kg) and were subsequently sacrificed at 36 h after injection. Liver biochemical indices were determined and liver tissue samples were stained using hematoxylin-eosin staining, followed by iTRAQ quantitative proteomics to identify potential underlying therapeutic mechanisms and biomarkers. The results suggested that the expression levels of alanine transaminase, aspartate aminotransferase, total bilirubin and direct bilirubin were reduced in the rats treated with melatonin. Histopathological observation indicated that melatonin was effective in the treatment of ANIT-induced cholestasis. iTRAQ proteomics results suggested that melatonin-mediated reduction in ANIT-induced cholestasis may be associated with enhanced antioxidant function and relieving abnormal fatty acid metabolism. According to pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes, the major metabolic pathways for the metabolism of melatonin are fatty acid degradation, the peroxisome proliferator-activated receptor signaling pathway, fatty acid metabolism, chemical carcinogenesis, carbon metabolism, pyruvate metabolism, fatty acid biosynthesis and retinol metabolism, as well as drug metabolism via cytochrome P450. Malate dehydrogenase 1 and glutathione S-transferase Yb-3 may serve as potential targets in the treatment of ANIT-induced cholestasis with melatonin.