Heterosis utilization is one of the effective ways to significantly increase crop yield， in which the creation and utilization of sterile lines plays a vital role in the breeding and production of crop hybrids. 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 design two gene editing targets to knock out the MS6 gene in soybean variety Williams82. The transgenic plants carrying edited MS6 alleles were subjected for phenotype observation， pollen and fertility identification， thus approving the function of this gene in regulating soybean pollen formation and male fertility. A new ms6 germplasm showing stable male sterility was obtained in soybean. This provides theoretical and technical support for further establishing the third generation soybean hybrid breeding technology system based on MS6 gene and realizing the efficient creation of strong heterosis hybrids.