Abstract: Fish exhibit a relatively low capacity for carbohydrate utilization. When fed a high-carbohydrate diet, fish exhibit increased hepatic glycogen content, which can disrupt normal liver metabolic functions. In recent years, investigating carbohydrate utilization and energy metabolism mechanisms across different fish species has become an active area of fish fish nutrition research. Under physiological conditions, blood glucose homeostasis is primarily maintained through the antagonistic actions of insulin and glucagon, with insulin being the most important hypoglycemic hormone in the body. Studies have found that endocrine mediators can influence the secretion and antagonistic balance between insulin and glucagon, ultimately regulating blood glucose levels by modulating glucose uptake, glycolysis, gluconeogenesis, as well as glycogen synthesis and breakdown. In mammals, spexin has been demonstrated to play significant roles in regulating glucose and lipid metabolism and alleviating insulin resistance. Spexin (SPX) is a polypeptide hormone composed of 14 amino acids, and two isoforms, SPX1 and SPX2, exist in fish. Our previous research has indicated that both SPX1 and SPX2 enhance insulin action in hepatocytes, thereby increasing hepatic insulin sensitivity. Furthermore, studies in fish have shown an association between spexin and the PI3K/Akt signaling pathway. Inhibition of PI3K or Akt attenuated insulin-mediated spx1 mRNA expression. Given that the PI3K/Akt pathway is a classical insulin signaling cascade, we hypothesized that spexin exerts its regulatory functions via this signaling axis. Using in vitro approaches, this study investigates the roles of SPX1 and SPX2 in regulating glucose metabolism via enhanced insulin-mediated PI3K/Akt signaling in the hepatocytes of grass carp (Ctenopharyngodon idella). Western blot analysis was used to examine the effects of spexin on the PI3K/Akt signaling pathway in the primary hepatocytes. Additionally, specific inhibitors of PI3K and Akt were employed to block this pathway, followed by quantitative real-time PCR to assess changes in insulin-mediated SPX1/SPX2 effects on glucose metabolism. Our results showed that insulin activated the PI3K/Akt signaling pathway, and the phosphorylation level of Akt protein peaked at 15 minutes after co-treatment of primary hepatocytes with insulin and 100 nM spexin (P < 0.05). SPX1 or SPX2 enhanced the activating effect of insulin on the PI3K/Akt signaling pathway, further elevating the mRNA expression levels of key glycolytic enzyme genes (gk, pk, and pfkla), glucose transporter 2 (glut2), and glycogen synthase gene (gys) (P < 0.05), and inhibiting the mRNA expression of key gluconeogenic enzyme genes (g6pase, pepck) and glycogen phosphorylase gene (pygl) (P < 0.05). These results indicated that SPX1 and SPX2 enhanced the activation of the PI3K/Akt signaling pathway activated by insulin, further promoting glycolysis and glycogen synthesis in the hepatocytes, and inhibiting gluconeogenesis and glycogenolysis pathways, which demonstrates that spexin enhance insulin-mediated regulation of glucose metabolism in the hepatocytes. This study clarified the indirect regulatory roles and mechanisms of spexin on glucose metabolism in the hepatocytes of grass carp, which provides a foundation for further in-depth understanding of the molecular mechanisms underlying the endocrine network regulating glucose metabolism in fish.