A proteomics approach was used to evaluate the effects of flooding stress on early symbiotic interaction between soybean roots and soil bacteria, Bradyrhizobium japonicum. Three-day-old soybean was inoculated with B. japonicum followed by flooding. The number of root hairs in seedlings, without or with flooding stress, was increased after 3 days of inoculation. Proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. Out of 219 protein spots, 14 and 8 proteins were increased and decreased, respectively, by inoculation under flooding compared with without flooding. These proteins were compared in untreated and flooded seedlings. Increased level of 6 proteins in flooded seedlings compared with untreated seedlings was suppressed by inoculation in seedlings under flooding. They were related to disease/defense, protein synthesis, energy, and metabolism. Differential abundance of glucan endo-1,3-beta-glucosidase, phosphoglycerate kinase, and triosephosphate isomerase, based on their localization in middle and tip of root, indicated that they might be related to increase in number of root hairs. These results suggest that disease/defense, energy, and metabolism-related proteins may be particularly subjected to regulation in flooded soybean seedlings, when inoculated with B. japonicum and that this regulation may lead to increase in number of root hair during early symbiotic differentiation.