Plant E2 Ubiquitin-conjugating enzymes regulate various biological pathways such as stress resistance, growth and development. Reports on its functions are more frequent in Arabidopsis thaliana, but relatively rare in soybean (Glycine max), which is one of the most important economic crops. In this study, a gene Glyma.12G161200, which may be related to the soybean cotyledon folding mutant, was cloned from soybean “Nanong 94-16”. Analysis of its sequence suggested that it encodes an E2 ubiquitin binding enzyme, so it was named as GmUBC1. Its coding region is 462 bp in length, which encodes a protein of 153 amino acids with a predicted molecular mass of 17.25 kDa and an isoelectric point of 6.74. The expression pattern of GmUBC1 in different tissues of soybean and its response patterns to different stresses and hormone treatments were analyzed by real-time PCR. The results showed that the gene was expressed at the highest level in mutant seeds at 40 days after flowering. Moreover, the expression of the GmUBC1 gene was down-regulated by the treatments of PEG, cold, JA and ABA, respectively. Subcellular localization analysis of GmUBC1 revealed that the protein was expressed in the whole cell. When GmUBC1 was ectopically expressed in Arabidopsis, the 1000-grain weight and total amino acid content of some transgenic lines were found to be significantly increased. Collectively, heterologous overexpression of GmUBC1 can regulate seed weights and amino acid contents, which may provide genetic resources for soybean quality improvement.