This study investigated the influence of berberine (BBR) supplementation in normal and high lipid diets on lipid metabolism and accumulation in black sea bream (Acanthopagrus schlegelii). Berberine was supplemented at 50 mg/kg to control (Con, 11.1% crude lipid) and high lipid (HL, 20.2% crude lipid) diets and named as ConB and HLB, respectively. After the 8-week feeding trial, fish body length and specific growth rate were significantly reduced by high lipid diets (P < 0.05). Muscle and whole-body crude lipid contents were significantly influenced by both BBR supplementation and dietary lipid level. Fish fed HLB diet had significantly lower serum triglyceride, low-density lipoprotein cholesterol contents, and alanine aminotransferase activity compared with the HL group. The HL group presented vast lipid accumulation in the liver, and hypertrophied hepatocytes along with large lipid droplets, and translocation of nuclear to the cell periphery. These abnormalities in black sea bream were alleviated in the HLB group. Berberine supplementation in high lipid diet significantly downregulated the hepatic expression levels of accα, srebp-1, 6pgd, g6pd and pparγ, whereas the lpl, hsl, and cpt1a expression levels were significantly upregulated. However, the expression levels of these genes showed opposite trends in muscle (except for pparγ). In conclusion, dietary BBR supplementation in high lipid diet reduced hepatic lipid accumulation by downregulating lipogenesis gene expression and upregulating lipolysis gene expression, and it increased muscle lipid contents with opposite trends of the mechanism observed in the liver.