Abstract: Heavy metal chromium (Cr) is one of China’s main soil pollutants and poses a great threat to its agricultural soils, especially in the Shanxi Province, where the soil Cr content is higher than the national average. A new millet (Setaria italica) variety, ‘Jingu 21’, has many advantages such as high quality, high yield and disease resistance. To investigate the changes and response mechanisms of fungal communities in agricultural soils during a period of Cr stress, we used soil (alkaline brown soil) planted with ‘Jingu 21’ as this study’s experimental material. Soil samples were taken before the introduction of Cr (CK), as well as 6 h (Cr_6 h) and 6 d (Cr_6 d) after 1 mmol L⁻¹ of Cr⁶⁺ was introduced to the soil. High-throughput sequencing and statistical analysis of the data were used to investigate the response mechanism, the soil fungal community establishment, and the functional prediction of fungal communities in ‘Jingu 21’ soils during the period of Cr stress. The spatial and temporal distribution patterns of soil fungal communities were investigated using the non-metric multidimensional scale analysis, the soil fungal community establishment driving mechanism was investigated by constructing an interspecific symbiotic network diagram and a neutral community model (NCM), and the changes in soil fungal community function were investigated using FUNGuild. The results revealed that the composition and structure of soil fungal communities differed significantly at the phylum and genus levels during the period of Cr stress, and the Shannon diversity index of the community decreased significantly (P<0.05) at the Cr_6 d stage (4.17 for CK, 3.81 for Cr_6 h, and 3.23 for Cr_6 d). The spatial and temporal distribution patterns of fungal communities were similar within the same Cr stress period and differed significantly across these periods. The fungal community establishment was dominated by stochastic process (beta NTI: −0.16 for CK, −0.71 for Cr_6 h, and −0.23 for Cr_6 d). The interspecific symbiotic network analysis revealed that the fungal species were mostly positively correlated with each other; the interspecific symbiotic network of the Cr_6 d stage had a higher number of edges, average degree, and average path length than those of the CK and Cr_6 h stages, indicating that the community was more stable in the Cr_6 d stage than in the CK and Cr_6 h stage. Gibberella, Fusarium, and Chrysosporium were the key genera in the network diagram. The NCM quantified the stochastic processes further indicated that the soil fungal community was widely distributed (migration rate m: 0.066 for CK, 0.132 for Cr_6 h, and 0.163 for Cr_6 d). The FUNGuild function prediction showed that the soil fungal community was dominated by pathogenic and saprophytic trophic types. In addition, the abundance of sensitive bacteria, such as Mortierella and Gibberella, decreased, and the abundance of resistant bacteria, such as Fusarium, increased, indicating that Cr stress may affected the abundance of sensitive and resistant fungi in the soil, with the highest abundance of Fusarium indicating possible soil contamination with pathogenic bacteria. Ultimately, the results of this study revealed that the fungal community in soil planted with ‘Jingu 21’ changed significantly during a period of Cr stress; the soil fungal community establishment was dominated by stochastic processes; the diffusion restrictions of the community gradually decreased; and the interspecific relationships were complex and primarily symbiotic. In conclusion, herein, we simulated the stress response of soil microbial communities to Cr stress by treating agricultural soils with Cr⁶⁺. In addition, we demonstrated the response mechanism of soil fungal communities during a period of Cr stress, which is an important consideration for the treatment and remediation of heavy metal-contaminated soils and the promotion of sustainable agricultural development.