Glycogen is the main energy storage material in mollusc, and the regulation of its metabolism is essential for the response against high temperature stress. In the present study, the alternation of lactic acid (LD) content, glycogen reserves, mRNA expression level of genes encoding glycogen metabolism enzymes and activities of glycogen metabolism enzymes in gills of Yesso scallop Patinopecten yessoensis after an acute high temperature treatment at 25°C were examined to understand the effect of high temperature on glycogen metabolism. The activity of T-ATPase in gills of scallops presented a gradual increase trend especially at 6h after the acute high temperature treatment (p<0.05). The glycogen reserves did not change significantly even there was a downward trend at 24h after the acute high temperature treatment (p>0.05). The mRNA transcripts of glycogen synthase (PyGCS) in gills of scallops decreased significantly at 1, 3, 6 and 12h (p<0.05), and recovered to normal level at 24h (p>0.05) after the acute high temperature treatment, while that of glycogen phosphorylase a (PyGPa) and phosphoenol pyruvate carboxy kinase (PyPEPCK) were both significantly down-regulated from 1h to 24h (p<0.05) after the acute high temperature treatment. The activity of PyGPa at 1, 12 and 24h and the content of LD at 3 and 24h in gills of scallops after the acute high temperature treatment both increased significantly (p<0.05). Furthermore, the mRNA transcripts of hexokinase (PyHK) and pyruvate kinase (PyPK) in gills of scallops increased significantly (p<0.05) after the acute high temperature treatment, and the response of PyHK was stronger. However, there was no significant difference on the activity of PyPK in gills of scallops between the experimental samples and the blank samples (p>0.05). In addition, the mRNA transcripts of citrate synthase (PyCS) in gills of scallops were significantly down-regulated at 6h and 12h (p<0.05), and finally returned to normal level at 24h (p>0.05) after the acute high temperature treatment. These results collectively indicated acute high temperature stress leaded the alternation of glycogen metabolism in the gills of Yesso scallop, glycogenesis, gluconeogenesis and TCA cycle were inhibited, and the glycolysis pathway of glycogen was enhanced to produce more energy for coping with environmental pressure.