Soybean (Glycine max) is an important crop for oil and protein resources worldwide. The molecular mechanism of abiotic stress response in soybean is largely unclear. We previously identified multiple stress-responsive WRKY genes from soybean. Here, we further characterized the roles of one of these genes, GmWRKY27, in abiotic stress tolerance using transgenic hairy root assay. GmWRKY27 expression was improved by various abiotic stresses. Overexpression and RNAi analysis demonstrate that GmWRKY27 improved salt and drought tolerance in transgenic soybean hairy roots. Measurement of physiological parameters including reactive oxygen species and proline contents also supported this conclusion. GmWRKY27 inhibits expression of a downstream gene GmNAC29 by binding to the W-boxes in its promoter region. The GmNAC29 is a negative effector of stress tolerance as revealed from the performance of transgenic hairy roots under stress. GmWRKY27 interacts with GmMYB174, which also suppresses GmNAC29 expression and enhances drought stress tolerance. The GmWRKY27 and GmMYB174 may have evolved to adapt to each other for binding to the neighbouring cis-elements in GmNAC29 promoter to co-reduce the promoter activity and gene expression. Our study discloses a valuable mechanism in soybean for stress response by two associated transcription factors. Manipulation of these genes should facilitate stress tolerance in soybean and other crops