Abstract: Carbohydrate response element binding protein (ChREBP) is a key transcription factor in the regulation of glucose and lipid metabolism in mammals. However, the function of ChREBP and its response to fatty acids in fish are still unknown. Therefore, in this study, we chose the large yellow croaker (Larimichthys crocea) as the research object, and firstly, this study cloned the sequence of the CDS region of the ChREBP gene in L. crocea. The results found that the full length of the CDS sequence of ChREBP in L. crocea is 2 808 bp, encoding 935 amino acids. Considering that the sequence of ChREBP has a certain degree of homology compared with the sequence of the mammal, ChREBP may have conserved functions in L. crocea. Real-time fluorescence quantitative PCR (RT-qPCR) was used to analyze the expression pattern of ChREBP gene in different tissues of L. crocea, and the results found that the mRNA levels of ChREBP gene were highest in the intestine of L. crocea, while it was also highly expressed in the liver. After 10 weeks of feeding trial, the mRNA levels of ChREBP gene were significantly lower (P < 0.05) in the liver of L. crocea fed palm oil, olive oil, linseed oil, and soya bean oil, compared with fish fed fish oil. After incubation of fish hepatocytes with different fatty acids for 12 h, the mRNA levels of ChREBP gene were significantly (P < 0.05) reduced in the palmitic acid (PA, C16:0) treatment, compared with the control treatment. However, the mRNA levels of ChREBP gene in linoleic acid (LA, C18:2n-6), linolenic acid (ALA, C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) treatments also showed a decreasing trend, but there was no significant difference (P > 0.05). The above results indicate that ChREBP of L. crocea could respond to the stimulation of different fatty acids at both in vivo and in vitro levels and might play an important role in the regulation of hepatic glucose and lipid metabolism. The above studies have further deepened the understanding of the mechanism of glucose and lipid metabolism regulation in fish, and provided a theoretical basis for improving the glucose and lipid metabolism disorders induced by the substitution of vegetable oils in L. crocea.