Citric acid is known to alleviate physical fatigue and improve athletic performance; however, the underlying mechanism is unknown. Therefore, we aimed to investigate the effect of citric acid administration on the expression of key glycolysis-related genes in the liver and skeletal muscle of rats. Eight-week-old male Sprague-Dawley rats were divided into two groups—control and citric acid groups. The rats were fasted overnight, after which distilled water or citric acid solution was administered intragastrically; after 150 min, the rats were sacrificed, and the liver and skeletal muscle were collected. In the skeletal muscle, namely soleus and gastrocnemius muscles, the phosphofructokinase and pyruvate kinase gene expression were significantly decreased by citric acid administration. In contrast, the glucokinase and pyruvate kinase gene expression in the liver was significantly increased. These results suggest that exogenous dietary citric acid suppresses glycolysis in the skeletal muscle at the transcription level.