Proline is a ubiquitous osmotic regulator that plays an important role in plant growth and development as well as signaling pathways in response to drought stress. P5CS, δ-OAT, P5CR, ProDH, P5CDH and ProT are the key enzymes that affect proline accumulation in plants. However, the members of the gene families related to proline accumulation in soybean remained to be systemically investigated. In this study, seven GmP5CS, two GmOAT, two GmP5CR, five GmProDH, three GmP5CDH and six GmProT genes were identified and found to be unevenly distributed on 12 of 20 soybean chromosomes, showing 16 pairs of fragment duplication events. The phylogenetic analysis showed that the soybean proline accumulation-related gene families were classified into different evolutionary branches, while within each branch a conservation on functional structure and motifs was observed. Analysis of cis-acting elements in these gene families revealed contained cis-acting elements that were reported associating with response to stress and plant hormones. Analysis of transcriptional profiles under drought stress treatment showed that the proline anabolism-related (GmP5CS, GmOAT, GmP5CR) gene family members were significantly up-regulated 24 hours post treatment, the proline catabolism-related gene family members (GmProDH, GmP5CDH) were significantly down-regulated, and the expression levels of proline transport-related gene family members (GmProT) were significantly up-regulated. Especially, GmP5CS5, GmOAT1, GmProT subgroup II and GmProDH3-5 genes may play key roles in proline accumulation under drought stress. Moreover, the P5CS and OAT activity of soybean seedlings, which was significantly increased along with the increase of drought stress time, positively correlated with proline accumulation. The ProDH activity was significantly decreased along with the increase of drought stress time, and was negatively correlated with the accumulation of proline. Collectively, this study provided information for further analyzing the functions of soybean proline accumulation-related family genes in response to drought stress.