Flooding stress causes growth inhibition and ultimately death in most crop species by limiting of energy production. To better understand plant responses to flooding stress, here, flooding-responsive proteins in the cotyledons of soybean were identified using a gel-free quantitative proteomic approach. One hundred forty six proteins were commonly observed in both control and flooding-stressed plants, and 19 were identified under only flooding stress conditions. The main functional categories were protein and development-related proteins. Protein-protein interaction analysis revealed that zincin-like metalloprotease and cupin family proteins were found to highly interact with other proteins under flooding stress. Plant stearoyl acyl-carrier protein, ascorbate peroxidase 1, and secretion-associated RAS superfamily 2 were down-regulated, whereas ferretin 1 was up-regulated at the transcription level. Notably, the levels of all corresponding proteins were decreased, indicating that mRNA translation to proteins is impaired under flooding conditions. Decreased levels of ferritin may lead to a strong deregulation of the expression of several metal transporter genes and over-accumulation of iron, which led to increased levels of reactive oxygen species, resulting to detoxification of these reactive species. Taken together, these results suggest that ferritin might have an essential role in protecting plant cells against oxidative damage under flooding conditions. BIOLOGICAL SIGNIFICANCE: This study reported the comparative proteomic analysis of cotyledon of soybean plants between non-flooding and flooding conditions using the gel-free quantitative techniques. Mass spectrometry analysis of the proteins from cotyledon resulted in the identification of a total of 165 proteins under flooding stress. These proteins were assigned to different functional categories, such as protein, development, stress, redox, and glycolysis. Therefore, this study provides not only the comparative proteomic analysis but also the molecular mechanism underlying the flooding responsive protein functions in the cotyledon.