Hedysari Radix, a significant Chinese herbal medicine from Northwest China's arid region, is renowned for its unique tonic effects in traditional Chinese medicine practices. This plant, a member of the Leguminosae family, forms a symbiotic relationship with nitrogen-fixing rhizobia. However, the Hedysarum polybotrys-rhizobium symbiotic system remains underexplored. The root nodule structure of H. polybotrys was examined using an optical microscope (OM). This examination revealed that its root nodules consist of meristematic zone, infection zone, nitrogen fixation zone, and senescence zone, arranged from top to bottom. This structure suggests that the root nodules of H. polybotrys belong to the indeterminate nodule category. In the fields of transmission electron microscopy (TEM) and fields emission scanning electron microscopy (FESEM), significant differences were observed between infected and un-infected cells. Rhizobium, identified via 16S rRNA technology and classified as the genus Mesorhizobium through phylogenetic analysis. Reinoculation of rhizobium into H. polybotrys seedlings resulted in nodule formation on the roots. Notably, inoculated plants exhibited a considerable increase in nodule number, leaf count, leaf length, aboveground height, aboveground fresh weight, root length, and root diameter compared to uninoculated controls, demonstrating that rhizobium inoculation enhances plant growth.