This study aimed to investigate the metabolic characteristics of hepatocytes under a high-glucose load in spotted sea bass (Lateolabrax maculatus). The liver cell line of L. maculatus was cultured, and two experimental groups were established. The cells from the control group were cultured in a medium containing 5 mmol/L glucose, whereas a medium containing 40 mmol/L glucose was used for the high-glucose group. After 48 h of culture, the cells and supernatants were collected and analyzed. Results showed that the glucose and glycogen contents in the high-glucose group were significantly increased (P＜0.05). The activities of hexokinase (HK), citrate synthase (CS), α-ketoglutarate dehydrogenase (α-KGDHC), isocitrate dehydrogenase (IDH), and succinate dehydrogenase (SDH) increased significantly (P＜0.05). These results indicate that a high-glucose load accelerates glycolysis and the tricarboxylic acid cycle in hepatocytes. In addition, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the supernatant increased with high-glucose culture (P＜0.05), indicating that high-glucose can cause hepatocyte damage. In the high-glucose group, the ATP content of hepatocytes significantly decreased (P＜0.05). The content of reactive oxygen species (ROS) and MDA was significantly increased (P＜0.05). Furthermore, the high-glucose culture upregulated the expressions of mitochondrial autophagy-related genes (pink, atg5, and mull) and down-regulated the expressions of mitochondrial biogenesis-related genes (nrf-1, pgc-1α, and pgc-1β) (P＜0.05). Sequencing of the mitochondrial genome showed that mitochondrial D-loop genes were mutated under a high-glucose load treatment. In conclusion, in vitro hepatocytes of sea bass showed similar physiological responses to those in vivo when they responded to high-glucose loads. Thus, in vitro hepatocytes could be used as a research platform to study glucose metabolism in fish. High-glucose cultures can lead to increased metabolic enzyme activity in hepatocytes, damaging hepatocytes and mitochondria. This is related to the oxidative stress caused by ROS accumulation. The high-glucose load upregulated the expression of mitochondrial autophagy-related genes and downregulated the expression of mitochondrial generation-related genes, decreasing the number of mitochondria in the cell. Mutations in the mitochondrial D-loop gene occur under a high-glucose load, affecting the structural stability of the mitochondria.