• Biocrust succession alters diazotrophic diversity and community compositions.
• Deterministic processes govern diazotrophic community assemblages.
• The TOC/TN ratio is a key factor driving diazotrophic community succession.
• Diazotrophic networks become less complex with biocrust succession.
The diazotrophic community in biological soil crusts (biocrusts) is the key supplier of nitrogen in dryland. To date, there is still limited information on how biocrust development influences the succession of diazotrophic community, and what are the most important factors mediating diazotrophic communities during biocrust succession. Using the high throughput nifH amplicon sequencing, the diazotrophs in soils at different developmental stages of biocrust were comparatively studied. The results evidenced the decrease of TOC/TN ratio and pH value with biocrust development. Nostoc and Scytonema were the most dominant diazotrophic genera at all biocrust stages, while Azospirillum and Bradyrhizobium were abundant only in bare soil. Diazotrophic co-occurrence networks tended to be less complex and less connected with biocrust succession. The soil TOC/TN ratio was the most dominant factor mediating diazotrophic diversity, community composition and assembly processes, while diazotrophic-diversity and NO3−-N/NH4+-N ratio were positively correlated with the nitrogenase activity during biocrust succession. This study provided novel understandings of nitrogen fixation and succession patterns of diazotrophic community, by showing the effects of biocrust succession on diazotrophic diversity, community composition, community assembly and co-occurrence networks, and recognizing TOC/TN ratio as the most dominant factor mediating diazotrophs during biocrust succession.