In order to further study the relationship between atp6 gene and Cytoplasm male sterility (CMS) of Bunching onion, the sequence of atp6 gene was obtained from male sterile line and maintainer line of Zhangqiu onion bunching using homologous cloning. The protein secondary structure and the expression of atp6 gene in different growth stages were compared. The results showed that there was one polymorphic locus (A/T) at the 171st base of the open reading frame of atp6 gene in CMS line and maintainer line, which changed the proportion of protein secondary structure slightly, but did not change the transmembrane structure of ATP6 protein. The expression of atp6 gene in roots and leaves/flowers at seedling, bolting, flowering and late flowering stage was detected by real-time fluorescence quantitative polymerase chain reaction. The results showed that the expression of atp6 gene in root showed a trend of increasing at first and then decreasing during the whole development of the plant, reaching the maximum at the flowering stage, and the expression of the maintainer line was 1.14 times higher than that of the CMS; The expression of atp6 gene in leaves/flowers decreased gradually during the whole growth period, and reached the highest level at the seedling stage, and the maintainer line was 1.96 times higher than that of the CMS.