Citric acid is recognized and utilized as a dietary supplement for its ability to eliminate fatigue. However, the molecular and cellular mechanism underlying this effect has not been elucidated. This study investigated the effect of intragastric citric acid administration on the energy metabolism and gene expression in the skeletal muscles of mice that had been fasted without exercise, using biochemical analyses of metabolic indicators and DNA microarrays. Expression of the genes involved in the glycolysis, tricarboxylic acid cycle and β-oxidation of fatty acids were not affected by citric acid administration. However, citric acid increased the level of blood glucose and expression of the phosphoenolpyruvate carboxykinase gene. These results indicate that citric acid ingestion may eliminate fatigue by promoting gluconeogenesis.