Soybean is a crop known to be susceptible to flooding and enhancing flooding tolerance may be a workable strategy to improve soybean production. To elucidate the effects of flooding on soybean metabolism, metabolite alterations in seedlings during flooding treatment were identified using capillary electrophoresis-mass spectrometry (CE/MS). The principal component analysis (PCA) of soybean seedlings revealed that the first component accounted for 62.2% of total variance, and the alteration of metabolites in control and flooding treatments appears to be separated by this component. Furthermore, comparison of the metabolic loading scores in the first component of PCA show that the significant metabolites for the first component were alanine (Ala), gamma-aminobutyric acid (GABA), citrate, fumarate and malate. Quantitative analysis revealed that the total soluble sugar content of seedlings in both control and flooding treatments had declined and was lower in the latter than the former. Phosphoenolpyruvate, pyruvate and lactate, which belong to glycolytic and fermentation pathways, increased transiently, but decreased 3 to 4 days after treatment. Citrate, 2-oxoglutarate, succinate, fumarate, Ala, and GABA, which are related to the TCA cycle and amino acid metabolism, accumulated during flooding treatment. These results suggest that metabolism associated with the TCA cycle, the Ala synthetic pathway, and the GABA shunt may be strongly influenced by flooding during soybean germination.