Here we describe its role as a transcriptional repressor which regulates ABA sensitivity. The truncated naturally mutated version, GmABAS1∆, was employed as a negative control. The nuclear localization of GmABAS1 was shown using the green fluorescent protein (GFP)-GmABAS1 fusion protein. The transcriptional repressor activity of GmABAS1 was demonstrated using yeast reporter systems. By overexpressing GmABAS1 in the transgenic soybean hairy root system, we identified the target gene of GmABAS1 to be Glyma.01G060300, an annotated ABI5 binding protein 3 (AFP3). Glyma.01G060300 and GmABAS1 exhibited reciprocal expression patterns under osmotic stress. The binding of GmABAS1 to the promoter of Glyma.01G060300 was demonstrated using electromobility shift assay (EMSA). The promoter binding and transcriptional repressor activities of GmABAS1 were further confirmed using a yeast promoter-binding reporter assay. The over-expression of GmABAS1 in Arabidopsis thaliana led to the down-regulation of AtAFP2 and enhanced sensitivity to ABA in the stomatal aperture and seed germination assays. This is the first time a 1R-subtype of MYB from soybean has been reported to be a transcriptional repressor involved in enhancing ABA sensitivity.