[Objective] To study the function of MCHK-3535 gene in symbiotic nitrogen fixation between Mesorhizobium huakuii 7653R and Astragalus sinicus. [Methods] We constructed the deletion mutant Δ3535, overexpression strains OV3535 and complementary strains C3535, followed by identifying symbiotic phenotypes, detecting the growth curve, swimming motility and biofilm formation Besides, we used qRT-PCR and promoter-GUS fusion vector to detect the expression characteristics of MCHK-3535 in the symbiotic process. [Results] Compared with the wild type 7653R, mutant Δ3535 grew faster, decreased motility, and increased biofilm formation, moreover, Δ3535 significantly increased the ability of nitrogen fixation and the fresh weight of plants, while the number and weight of root nodules were not affected, in addition, the complementary strain C3535 partially compensated to the wild-type phenotype, whereas OV3535 had no significant difference in these aspects. The results of qRT-PCR and the promoter-GUS reporter system showed that MCHK-3535 expressed in the infection zone, transitional zone and nitrogen fixation zone of the nodules, and the gene expressed lasted for the entire period of nitrogen fixation. [Conclusion] MCHK-3535 functions in the symbiotic nitrogen fixation process and may participates in the normal development of nodules and negatively regulates nitrogenase activity.