In plants, the terminal step of cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OAS-TL, EC 184.108.40.206). However, to date, there is limited information on those genes involved in this pathway in soybean. In this study, six soybean OAS-TL-like genes were obtained and designated as GmOAS-TL1, 2, 3, 4, 6, and 7. Phylogenetic analysis indicated that GmOAS-TL1, 2, 6, and 7 were similar and showed homology to cytosolic isoforms, while GmOAS-TL3 and 4 were homologous to β-cyanoalanine synthase (CAS, EC 220.127.116.11)-like and a plastidic isoform, respectively. Both genes seemed to belong to a small gene family as confirmed by Southern blot analysis. Expression of GmOAS-TL1, 3, 4, and 6 in the Escherichia coli cys- auxotroph resulted in bacterial growth and exhibited high enzymatic activity. Reverse transcription polymerase chain reaction analysis indicated that varying levels of expression were detected in different tissues and with distinct profiles. During the stage of seed-filling, expression patterns showed a cooperative trend that corresponded with total OAS-TL enzymatic activity. However, the enzymatic activity was highly negatively correlated with cysteine content in the developing seeds. These results suggest that cysteine synthesis in soybean might be an operation of multiple OAS-TL genes with homeostasis mechanisms for self-regulation. This finding should enhance our understanding of the molecular basis of soybean cysteine biosynthesis.