Gel-based and gel-free proteomics techniques were used to investigate early responses to flooding stress in the roots and hypocotyls of soybean seedlings. Proteins from 2-day-old soybean seedlings flooded for 12 h were extracted and analyzed. Two mass-spectroscopy-based proteomics analyses, two-dimensional fluorescence difference gel electrophoresis, and nanoliquid chromatography identified 32 from 17 spots and 81 proteins, respectively, as responsive to flooding stress. On the basis of the number and function of proteins identified, glycolysis and fermentation enzymes and inducers of heat shock proteins were key elements in the early responses to flooding stress. Analysis of enzyme activities and carbohydrate contents in flooded seedlings showed that glucose degradation and sucrose accumulation accelerated during flooding due to activation of glycolysis and down-regulation of sucrose degrading enzymes. Additionally, the methylglyoxal pathway, which is detoxification system linked to glycolysis, was up-regulated. Furthermore, two-dimensional polyacrylamide gel electrophoresis-based phosphoproteomics analysis showed that proteins involved in protein folding and synthesis were dephosphorylated under flooding conditions. These results suggest that translational and post-translational control during flooding possibly induces an imbalance in the expression of proteins involved in several metabolic pathways including carbohydrate metabolism that might cause flooding injury of soybean seedlings.