Abstract Soybean [Glycine max (L.) Merr.] is an important crop providing vegetable oils and proteins. Increasing demand on soy products heightens the urgency of soybean yield improvement. Hybrid breeding with male sterility system is an effective method to improve crop production. Cloning of genic male sterile (GMS) gene combined with biotechnology method can contribute to constructing GMS-based hybrid Seed Production Technology (SPT) to promote soybean performance and yield. In this research, we identified a soybean GMS locus, GmMS6, by combining bulked segregant analysis (BSA)-sequencing and map-based cloning technology. GmMS6 encodes an R2R3 MYB transcription factor, whose mutant allele in ms6 (Ames1) harbors a single nucleotide polymorphism (SNP) substitution, leading to the 76th Leucine to Histidine change in the DNA binding domain. Phylogenetic analysis demonstrates GmMS6 is a homolog of Tapetal Development and Function 1 (TDF1)/MYB35 that is an anther development key factor co-evolved with angiosperm. It has a recently duplicated homolog GmMS6LIKE (GmMS6L), both of which can rescue the male fertility of Arabidopsis homologous mutant attdf1 while GmMS6L76H cannot, denoting that both proteins are functional and L76 is a critical residue for TDF1’s function. However, compared to anther specific expressed GmMS6, GmMS6L is constitutively expressed at a very low level, explaining deficiency of GmMS6 alone causes pollen abortion. Moreover, the expression levels of major regulatory and structural genes for anther development are significantly decreased in ms6, unveiling that GmMS6 is a core transcription factor regulating soybean anther development.