Fish are poor users of dietary carbohydrates and considered to be glucose intolerant, and activating AMP‐activated protein kinase (AMPK) by 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) has been shown to stimulate glucose uptake and utilization in skeletal muscle cells of rainbow trout. However, the effects of activating AMPK by dietary AICAR in the piscine model remain largely unexplored. In the present study, grass carp were fed purified diets including AICAR at a level of 0 or 200 mg/kg diet to chronically activate AMPK for 8 weeks, and the dietary effects on phenotypic and molecular responses related to glucose and lipid metabolism were examined. The results showed that activating AMPK by dietary AICAR significantly reduced fish growth performance, serum glucose and triacylglycerol contents, and liver lipid contents, while it increased hepatic glycogen contents (p 0.05). The results of AMPK signalling pathway in both the liver and muscle show that dietary AICAR increased the phosphorylation of AMPKα and acetyl‐CoA carboxylase (ACC; p < 0.05), while inhibiting the phosphorylation of sterol regulatory element‐binding transcription factor‐1(SREBP‐1) and target of rapamycin (p < 0.05). The activation of AMPK reduced the mRNA expression of lipogenesis enzymes in the liver and muscle (p < 0.05) and increased mRNA expression of gluconeogenesis enzymes in the liver and glycolysis enzymes in the heart (p < 0.05). Our results demonstrate that activating AMPK by AICAR reduces serum glucose and inhibits activities of ACC and SREBP‐1 to reduce lipogenesis in the liver and muscle, and AMPK could be used as an active target for its metabolic syndromes of grass carp.