Soybean [Glycine max (L.) Merr.] is an exceptionally rich in isoflavones, and these compounds attach to oestrogen receptors in the human body, lessening the risk of breast cancer and effectively alleviating menopausal syndrome symptoms. Uncovering the molecular mechanisms that regulate soybean isoflavone accumulation is crucial for enhancing the production of these compounds. In this study, we combined bulk segregant analysis sequencing (BSA-seq) and a genome-wide association study (GWAS) to discover a novel R2R3-MYB family gene, GmMYB77, that regulates isoflavone accumulation in soybean. Using the soybean hairy root transient expression system, we verified that GmMYB77 inhibits isoflavone accumulation. Furthermore, knocking out GmMYB77 significantly increased total isoflavone (TIF) content, particularly malonylglycitin, while its overexpression resulted in a notable decrease in contents of malonylglycitin and TIF. We found that GmMYB77 can directly binds the core sequence GGT and suppresses the expression of the key isoflavone biosynthesis genes Isoflavone synthase 1 (GmIFS1), Isoflavone synthase 2 (GmIFS2), Chalcone synthase 7 (GmCHS7) and Chalcone synthase 8 (GmCHS8) by using dual-luciferase assays, electrophoretic mobility shift assays and yeast one-hybrid experiments. Natural variations in the promoter region of GmMYB77 affect its expression, thereby regulating the malonylglycitin and TIF contents. Hap-P2, an elite haplotype, plays a pivotal role in soybean breeding for substantially enhanced isoflavone content. These findings enhance our understanding of the genes influencing soybean isoflavone content and provide a valuable genetic resource for molecular breeding efforts in the future.