Glutaredoxins (Grx) are redoxin family proteins that reduce disulfifides and mixed disulfifides between glutathione and proteins. Rhizobium leguminosarum 3841 contains three genes coding for glutaredoxins: RL4289 (grxA) codes for a dithiolic glutaredoxin, RL2615 (grxB) codes for a monothiol glutaredoxin, while RL4261 (grxC) codes for a glutaredoxin-like NrdH protein. In this study, we have generated mutants which have interrupted one, two, or three glutaredoxin genes. These mutants had no different growth phenotypes from the wild type RL3841. Mutation of grxC did not affect R. leguminosarum antioxidant or symbiotic capacities, while grxA-derived or grxB-derived mutants decreased the antioxidant and fixation nitrogen capacity. grxA-derived mutants was severely impaired in there rhizosphere colonization, and formed maller nodules with defects of bacteroid differentiation. whereas nodules induced by grxB-derived mutants contained abnormally thick cortexs and prematurely senescent bacteroids. Surprisingly the grx triple mutant presented an enhanced defect in antioxidant and symbiotic capacities of R. leguminosarum. LC-MS/MS analysis quantitative proteomics techniques were employed to compare differential grx triple mutant root bacteroids in response to the wild type infection. 137 differentially expressed proteins were identified including 56 up-regulated and 81 down-regulated proteins. By sorting the identified proteins according to metabolic function, twenty-eight proteins were involved in transporter activity, twenty proteins were related to stress response and virulence, and sixteen proteins were related to amino acid metabolism. Overall, R. leguminosarum glutaredoxins play an important role in root nodule symbiosis by involving the functions of anti-oxidant defenses, Fe-S cluster assembly, and control of bacteroid differentiation and senescence.