Compared with mammals, fish have a limited capability to utilize carbohydrates, thus generally suffering from metabolic disorders when offered carbohydrate-enriched diets. As a synthetic liposoluble derivative of vitamin B(1), benfotiamine can alleviate the carbohydrate overload-induced mitochondrial dysfunction in fish, but the potential mechanisms have not been well explored. The present research was performed to unveil the molecular pathways through which benfotiamine benefits the mitochondrial function of a carp species Megalobrama amblycephala, which often exhibits metabolic disturbances. First, a control (C, 30% carbohydrate) group, a high-carbohydrate (HC, 43% carbohydrate) group, and a HC incorporating benfotiamine (1.425 mg/kg) group were conducted, respectively, in a 12-week feeding trial. Then, two in vitro studies were performed by using primary hepatocytes. In the first one, a media treatment, a high-glucose (HG) treatment, and a HG incorporating benfotiamine were designated, respectively. In the second one, a media group, a vehicle group, a HG group, and a HG + BL-918 (the agonist of UNC-51-like kinase 1 [ULK1]) group were adopted, respectively. The results indicated that HC/HG treatment resulted in mitophagy disorder by downregulating the phosphorylation of AMPK and ULK1 and the contents of proteins involved in the PTEN-induced putative kinase protein 1 (PINK1)-Parkin pathway. Mitochondrial dysfunction was also observed, as was indicative of the reduced activities of mitochondrial complex I, III, and SDH. However, benfotiamine treatment increased the contents of P-AMPK, P-ULK1, and the PINK1-Parkin pathway-related proteins as well as mitochondrial complex activities. In conclusion, benfotiamine could trigger the ULK1-mediated mitophagy to ameliorate the carbohydrate overload-induced mitochondrial dysfunction in fish.