In contrast to the traditional hybrid breeding technology, the third generation crop hybrid breeding technology based on the nuclear male sterility gene has layers of advantages such as safety on seed production, flexibility on bi-parental combinations, and stability on hybrid fertility. This technology has been widely used in maize, rice and other crops, and provides a possibility in future use of soybean heterosis. We previously mapped by positional cloning approach a nucleic male sterility gene MS6 that encodes an R2R3-MYB transcription factor in soybean. In this study, we deployed CRISPR/Cas9 gene editing technology to knock out the MS6 gene in soybean. The transgenic plants carrying edited MS6 alleles were subjected for phenotype observation, pollen and fertility identification, thus approving the causal mechanism of this gene. A new germplasm showing stable male sterility was obtained. This provides theoretical and technical support for further establishing the third generation soybean hybrid breeding technology system based on MS6 gene.