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This study tested the effect of berberine on autophagy-related protein of Megalobrama amblycephalafed with high fat diet under different feeding modes. The full-length complementary DNA (cDNA) of microtubule-associated protein 1-light chain 3B (LC3B) was 1871 bp with an open reading frame of 378 bp encoding 125 amino acids. High homology at nucleotide and amino acid sequences to carp LC3B was revealed though sequence analysis. LC3B was mainly (P <0.05) expressed in hepatopancreas but lower in several peripheral tissues, including gill, intestine, kidney, and spleen. The fish (average initial weight 4.70 ± 0.02 g) were fed with eight experiment diets containing two lipid levels (5 and 10%) or four berberine-feeding modes (without berberine, supplementing 50 mg/kg berberine at 2-week intervals, 4-week intervals, or continuous) for 8 weeks. The results showed that the numbers of autophagosomes and hepatopancreas LC3B messenger RNA (mRNA) expression levels were significantly (P <0.05) affected both by dietary lipid level and beberine feeding mode, and the highest (P< 0.05) numbers of autophagosomes and LC3B expression levels were observed in fish at berberine continuous feeding mode groups. The findings may provide the molecular mechanisms underlying lipid metabolism and immune effect of berberine, which was associated with enhanced autophagy in fish.