Bacillus velezensis S141, a plant growth-promoting rhizobacteria (PGPR), was isolated from a soybean field in Thailand. Previous studies demonstrated that S141 enhanced soybean growth, stimulating nodulation for symbiotic nitrogen fixation with soybean root nodul e bact eri a, i ncl udi ng Bradyrhizobium diazoefficience USDA110. Isoflavone glycosides are produced in soybean roots and hydro-lyzed into their aglycones, triggering nodulation. This study revealed that S141 efficiently hydrolyzed two isoflavone glycosides in soybean roots (daidzin and genistin) to their aglycones (daidzein and genistein, respectively). However, S141, Bacillus subtilis 168, NCIB3610, and B. velezensis FZB42 hydrolyzed isoflavone glucosides into aglycones. A BLASTp search suggested that S141 and the other three strai ns shared four genes encodi ng β-glucosidases corresponding to bglA, bglC, bglH, and gmuD in B. subtilis 168. The gene inactivation analysis of B. subtilis 168 revealed that bglC encoded the major β-glucosidase, contributing about half of the total activity to hydrolyze isoflavone glycosides and that bglA, bglH, and gmuD, all barely commit-ted to the hydrolysis of isoflavone glycosides. Thus, an unknown β-glucosidase exists, and our genetic knowledge of β-glucosidases was insufficient to eval-uate the ability to hydrolyze isoflavone glycosides. Nevertheless, S141 could predominate in the soybean rhizosphere, releasing isoflavone aglycones to enhance soybean nodulation. © 2023 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation.