Flooding negatively affects the soybean growth; however, silver nanoparticles enhanced the growth under stress. To study the effects of silver nanoparticles on soybean under flooding stress in time-course manner, a gel-free proteomic technique was used. The morphological analysis of early-stage soybean exposed to flooding with silver nanoparticles of various sizes and concentrations revealed enhanced seedling growth by treatment with 15 nm silver nanoparticles at 2 ppm concentration. Differentially changed 107 root proteins were predominantly associated with stress, signaling, and cell metabolism. Hierarchical clustering divided these proteins into 3 clusters. Based on cluster analysis, the abundances of glyoxalase II 3 and fermentation related proteins, which were pyruvate decarboxylase 2 and alcohol dehydrogenase 1, were time-dependently increased under flooding stress, but decreased in response to silver nanoparticles. Six enzymes involved in metabolic pathways were analyzed at the transcriptional level. The alcohol dehydrogenase 1 and pyruvate decarboxylase 2 genes were up-regulated under flooding stress while down-regulated in response to silver nanoparticles. Moreover, comparatively low transcript level of glyoxalase II 3 under silver nanoparticles treatment implies that less cytotoxic by-products of glycolysis are produced in silver nanoparticles exposed soybeans as compared to flooded soybean. These results suggest that the silver nanoparticles treated soybeans have experienced less oxygen-deprivation stress, which might be the key factor for better growth performance of silver nanoparticles treated soybeans under flooding stress.