Abstract: Carnivorous fish exhibit a limited capacity to utilize dietary carbohydrates, and excessive carbohydrate intake can impair the growth performance and health. However, current research on carbohydrate metabolism in fish primarily focuses on metabolic pathways such as gluconeogenesis and glycolysis, with insufficient attention given to glucose transport mechanism. In mammals, AMPK can influence the intracellular translocation of GLUT4, thereby affecting glucose uptake, but its mechanism in carnivorous fish remains unclear. Therefore, this experiment utilized both in vivo and in vitro models to investigate the regulatory role of the AMPKα1/FNDC5 axis in glucose absorption in the skeletal muscle of Monopterus albus. Tissue distribution analysis indicated that fndc5, ampkα1, ampkα2, and tbc1d1 were expressed in the spleen, adipose tissue, kidney, liver, dorsal muscle, hindgut and brain of M. albus. The highest expression of ampkα1 was observed in the brain, hindgut and liver, while the highest expression of fndc5, ampkα2, and tbc1d1 was observed in the brain. Subcellular localization results showed that AMPKα1, TBC1D1, and FNDC5 were mainly distributed in the cytoplasm of HEK-293T cells. Injection of an ampkα1 overexpression plasmid (ADV-CMV-AMPKα-EGFP-pA) into the dorsal muscle of M. albus significantly decreased serum glucose and insulin levels, while significantly increasing serum irisin levels. The relative mRNA expression levels of ampkα1, tbc1d1, glut4, and fndc5 in muscle were also significantly upregulated. Further verification in HEK-293T cells revealed that overexpression of ampkα1 for 12 h significantly upregulated the relative mRNA expression level of fndc5; overexpression of ampkα1 for 24 h significantly upregulated the relative mRNA expression levels of fndc5, tbc1d1 and glut4. Additionally, overexpression of fndc5 for 12 h in HEK-293T cells significantly upregulated the relative mRNA expression level of ampkα2 and significantly downregulated the relative mRNA expression level of tbc1d1; while overexpression of fndc5 for 24 h significantly upregulated the relative mRNA expression level of glut4 and significantly downregulated the relative mRNA expression level of ampkα1. In conclusion, the AMPKα1/FNDC5 axis may represent a potential mechanism for enhancing glucose absorption in M. albus skeletal muscle. This study has clarified the regulatory role of the AMPKα1/FNDC5 axis in glucose absorption in the skeletal muscles of M. albus, providing a theoretical basis for regulating carbohydrate metabolism and formulating feed for carnivorous freshwater fish.