The receptor for activated C kinase 1 (RACK1) belongs to a protein subfamily that contains a tryptophan-aspartic acid-domain (WD) repeat structure. In this study, a soybean RACK1 gene (GmRACK1) was cloned. The amino acid sequence of GmRACK1 had seven WD repeats, which contained typical glycine-histidine (GH) and WD dipeptides. Tissue-specific expression showed that GmRACK1 is expressed at differential levels in all examined tissues and strongly down-regulatd by abscisic acid (ABA) and abiotic stress. Subcellular localization suggested that GmRACK1 is located in the plasma membrane, cytoplasm, and nucleus. In response to drought and salt stress, the GmRACK1-RNAi lines showed significantly higher dry weight of whole plants, chlorophyll content, and survival rate of soybean seedlings than wild-type and homozygous (OE) lines. GmRACK1-RNAi plants were observed to be more sensitive to ABA-mediated seed germination and root growth. Furthermore, we found that the level of ABA and transcript levels of stress-responsive genes were clearly up-regulated under drought and salt stress conditions in GmRACK1-RNAi plants. Consistent with the accumulation of reactive oxygen species (ROS), elevated electrolyte leakage and malondialdehyde, and lower expression of ROS-scavenging genes were found in the GmRACK1-OE lines. The GmRACK RNAi lines exhibited increased tolerance to drought and salt stress compared with the wild-type and OE lines. In addition, yeast two-hybrid assays showed that GmRACK1 could interact with WNK8, eIF6, and RbohC-N. These results therefore indicate that GmRACK1 responds to drought and salt stress through ABA signaling and the regulation of cellular ROS production in plants.