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Graphene has found important applications in various areas and hundred tons of graphene materials are annually produced. It is crucial to investigate both the negative and positive environmental effects of graphene materials to ensure the safe applications and develop environmental applications. In this study, we reported the stimulating effects of reduced graphene oxide (RGO) to nitrogen-fixing bacterium Azotobacter chroococcum. RGO stimulated the cell growth of A. chroococcum at 0.010-0.500 mg/mL according to the growth curves and the colony-forming unit (CFU) increases. RGO wrapped over the A. chroococcum cells without inducing ultrastructural changes. RGO decreased the leakage of cell membrane, but slight oxidative stress was observed in A. chroococcum. RGO promoted the nitrogen fixation activity of A. chroococcum at 0.5 mg/mL according to both isotope dilution method and acetylene reduction activity measurements. Consequently, the increases of soil nitrogen contents were evidenced, in particular about 30% increase of organic nitrogen occurred at 0.5 mg/mL of RGO. In addition, RGO might possibly benefit the plant growth through enhancing the indoleacetic acid production of A. chroococcum. These results highlighted the positive environmental effects of graphene materials to nitrogen-fixing bacteria in nitrogen cycle., (Copyright © 2022 Elsevier Ltd. All rights reserved.)