Aluminum oxide (Al2O3) nanoparticles are used in agricultural products and cause various adverse growth effects on different plant species. To study the effects of Al2O3 nanoparticles on soybean under flooding stress, a gel-free proteomic technique was used. Morphological analysis revealed that treatment with 50 ppm Al2O3 nanoparticles under flooding stress enhanced soybean growth compared to ZnO and Ag nanoparticles. A total of 172 common proteins that significantly changed in abundance among control, flooding-stressed, and flooding-stressed soybean treated with Al2O3 nanoparticles were mainly related to energy metabolism. Under Al2O3 nanoparticles the energy metabolism was decreased compared to flooding stress. Hierarchical clustering divided identified proteins into four clusters, with proteins related to glycolysis exhibiting the greatest changes in abundance. Al2O3 nanoparticle-responsive proteins were predominantly related to protein synthesis/degradation, glycolysis, and lipid metabolism. mRNA expression analysis of Al2O3 nanoparticle-responsive proteins that displayed a 5-fold change in abundance revealed that NmrA-like negative transcriptional regulator was up-regulated, and flavodoxin-like quinone reductase was down-regulated. Moreover, cell death in root including hypocotyl was less evident in flooding-stressed with Al2O3 nanoparticles compared to flooding-treated soybean. These results suggest that Al2O3 nanoparticles might promote the growth of soybean under flooding stress by regulating energy metabolism and cell death.