Glucose is used as one of the energy sources in many organs. Under feeding conditions, glucose can be obtained from dietary carbohydrate. On the other hand, when external supply of energy is interrupted, such as fasting, carbohydrate preserved in liver and glycogenic precursor derived from other organs are used to maintain blood glucose levels. Glycerol and glycogenic amino acid derived from adipocyte and skeletal muscle, respectively, are utilized as glycogenic precursor. But, it hasn't become clear which glycogenic precursor is more important in gluconeogenesis. The recent study revealed that Metformin, therapeutics for treating type 2 diabetes, depressed gluconeogenesis from glycerol by inhibition of glycerol‐3‐phosphate dehydrogenase (GPD) 2. This result suggests that glycerol have a potent impact on gluconeogenesis. In this study, we focused attention on GPD1, which is the enzyme that related gluconeogenesis using glycerol, and examined the roles of GPD1 in gluconeogenesis and importance of glycerol as glycogenic precursor. Using GPD1 null mutant model BALB/cHeA mice (HeA mice), we measured the change of blood glucose levels under fasting conditions and gene expressions related to gluconeogenesis in liver. BALB/cBy mice (By mice) were used as a control. In HeA mice, blood glucose levels at 1 to 4 hour after fasting were significantly higher than the By mice. The hepatic gene expressions involved in gluconeogenesis and aminotransferase such as glucose‐6‐phosphatase and alanine amino transferase were also higher than the By mice. Moreover, we examined availability of the glycogenic amino acid by alanine tolerance test. At 30 min after alanine administration, blood glucose level was significantly higher in the HeA mice. In short, these data suggests that lacking of GPD1 maintained blood glucose levels under fasting state by enhancing gluconeogenesis from glycogenic amino acid and release of glucose into the blood stream.Support or Funding InformationThis study was supported by the Council for Science, Technology and Innovation (CSTI), Cross‐ministerial Strategic Innovation Promotion Program (SIP, No.14533567), and “Technologies for creating next‐generation agriculture, forestry and fisheries” (funding agency: Bio‐oriented Technology Research Advancement Institution, NARO), Grants‐in‐Aid for Scientific Research (KAKENHI, No. 15J10165, 15K00827) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT, Tokyo), and University of Shizuoka Grant for Scientific and Educational Research.