Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar-dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1(a) allele encodes the xylosyltransferase UGT73F4, whereas Sg-1(b) encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1(a) and Gly-138 in Sg-1(b) proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-1(0) is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.