WRKY transcriptional factors play important roles in response to various abiotic stresses. In this study, we demonstrate that inserting a copy of soybean WRKY54 driven by a constitutive promoter of CMP (cestrum yellow leaf curling virus) or a drought induced promoter of RD29a, improves drought tolerance in transgenic soybean plants. GmWRKY54 is a transcriptional activator and regulates expressions of large numbers of stress-related genes. Using a GO enrichment toolkit, we found that GmWRKY54 up-regulated GO terms related to transcriptional regulator and gene expression and down-regulated GO terms related to photosynthesis and membrane. Using a co-expression network analysis, we find that dozens of genes are negatively co-expressing with photosynthetic genes. These genes are related to ABA signaling pathway, Ca2+ signaling pathway and transcriptional factors. Transgenic plants confer drought tolerance through improving stomatal closure, reducing transpiration rate and reducing membrane damage during water deficit. GmWRKY54 activates expression of these ABA or Ca2+ signaling-related genes, which finally regulates stomatal aperture. GmWRKY54 binds to the promoter regions of genes encoding an ABA receptor (PYL8-1), two calcium dependent protein kinase (CPK3-1 and CPK3-2), a CBL (Calcineurin B-like protein) interacting protein kinase (CIPK11-2), a SnRK family protein kinase and a ferritin, suggesting that they are the direct targets of GmWRKY54. Our study reveals that GmWRKY54 significantly improves drought tolerance in soybean and regulates expression of large numbers of drought-related genes, suggesting that GmWRKy54 maybe a master regulator of drought response. Overall design: Three transgenic soybean samples, A004A, A005B and B010C and the control plants (null) were chosen for RNA-seq. Both well-water samples and dehydrated samples were analyzed.