Fungal communities play a major role as decomposers in the Earth's ecosystems. Their community-level responses to elevated CO 2 (eCO 2 ), one of the major global change factors impacting ecosystems, are not well understood. Using 28S rRNA gene amplicon sequencing and co-occurrence ecological network approaches, we analyzed the response of soil fungal communities in the BioCON (biodiversity, CO 2 , and N deposition) experimental site in Minnesota, USA, in which a grassland ecosystem has been exposed to eCO 2 for 12 years. Long-term eCO 2 did not significantly change the overall fungal community structure and species richness, but significantly increased community evenness and diversity. The relative abundances of 119 operational taxonomic units (OTU; ϳ27% of the total captured sequences) were changed significantly. Significantly changed OTU under eCO 2 were associated with decreased overall relative abundance of Ascomycota, but increased relative abundance of Basidiomycota. Cooccurrence ecological network analysis indicated that eCO 2 increased fungal community network complexity, as evidenced by higher intermodular and intramodular connectivity and shorter geodesic distance. In contrast, decreased connections for dominant fungal species were observed in the eCO 2 network. Community reassembly of unrelated fungal species into highly connected dense modules was observed. Such changes in the co-occurrence network topology were significantly associated with altered soil and plant properties under eCO 2 , especially with increased plant biomass and NH 4 ؉ availability. This study provided novel insights into how eCO 2 shapes soil fungal communities in grassland ecosystems.