Biological nitrogen fixation is a major pathway in ecosystems. This study investigated the effects of adding Cu at different levels (0, 200, and 2000 mg kg(-1)) on the diazotroph community during swine manure composting. Quantitative PCR and high-throughput sequencing were used to analyze the abundances of diazotrophs and the community composition based on the nifH gene. The nifH gene copy number was relatively high in the early stage of composting and Cu had a significant inhibitory effect on the nifH copy number. Furthermore, Cu decreased the diversity of nifH and changed the microbial community structure in the early stage. The nifH genes from members of Firmicutes and Clostridium were most abundant. Co-occurrence ecological network analysis showed that the Cu treatments affected the co-occurrence patterns of diazotroph communities and reduced the associations between different diazotrophs. Interestingly, Cu may weaken symbiotic diazotrophic interactions and enhance the roles of free-living diazotrophs.