Cyhalofop-butyl is a highly effective aryloxyphenoxypropionate herbicide and widely used for weed control in paddy fields. With the increasing residue of herbicides, it poses a threat to the survival of aquatic organisms. Here, we evaluated the effect of cyhalofop-butyl on zebrafish to explore its potential hepatotoxic mechanism. The results showed that cyhalofop-butyl (0.1, 0.2 and 0.4 mg/L) induced hepatocyte degeneration, vacuolation and necrosis of larvae after embryonic exposure for 4 days and caused liver atrophy after exposure for 5 days. Meanwhile, the activities of enzymes related to liver function named alanine transaminase (ALT), aspartate transaminase (AST) were significantly increased by 0.2 mg/L cyhalofop-butyl and higher. And the contents of triglyceride (TG) involved in lipid metabolism was significantly decreased by 0.4 mg/L cyhalofop-buty. The effects of cyhalofop-butyl on zebrafish larvae were further demonstrated by GO (Gene Ontology) and KEGG pathway analysis. Cyhalofop-butyl (0.1, 0.2, 0.4 mg/L) altered the expression of 116, 154, 397 genes in liver，these genes are mainly enriched in metabolism (such as lipid metabolism, amino acid metabolism), immune system (Toll-like receptor signaling pathway) and endocrine system (PPAR signaling pathway). Furthermore, the expression of key genes related to liver development combined with RNA-Seq results, indicated that cyhalofop-butyl might damage the liver development of zebrafish larvae and cause metabolic disorders. To sum up, our research results reveal the physiological and molecular responses of zebrafish liver to cyhalofop-butyl and provide new insights for further studying the mechanism of cyhalofop-butyl toxicity to aquatic organisms.