<jats:title>Abstract</jats:title>
          <jats:sec>
            <jats:title>Background</jats:title>
            <jats:p>Legume-based rotation modulates soil microbial communities, which are critical to soil health and sustainable agriculture. However, current researches primarily focus on the effects of legume-based rotation on the bacterial community, leaving eukaryotic responses to this rotation poorly characterized.</jats:p>
          </jats:sec>
          <jats:sec>
            <jats:title>Methods</jats:title>
            <jats:p>We analyzed soil eukaryotic communities via 18 S rRNA sequencing in a Mollisol region under maize monoculture versus maize-soybean rotation, integrating soil physicochemical data and network analysis to elucidate rotation-driven eukaryotic assembly patterns.</jats:p>
          </jats:sec>
          <jats:sec>
            <jats:title>Results</jats:title>
            <jats:p>Maize-soybean rotation increased fungal and protistan richness by 53.04% and 23.80%, respectively, compared to those in the maize continuous cropping system, while the nematode was not sensitive. Maize-soybean rotation significantly altered fungal and protistan community compositions, increasing the relative abundances of Ascomycota (from 35.79 to 58.51%) and Alveolata (from 13.80 to 18.06%), respectively. Deterministic processes governed these shifts, with soil total carbon driving fungal richness and moisture regulating protistan β-diversity. Reduced environmental filtering under maize-soybean rotation enhanced the richness and network complexity of fungi and protists. Structural equation modeling (SEM) analysis revealed that the maize-soybean rotation improved soil multifunctionality by increasing fungal richness and complicating protistan community structure.</jats:p>
          </jats:sec>
          <jats:sec>
            <jats:title>Conclusions</jats:title>
            <jats:p>Our study highlights the pivotal role of fungal diversity and protistan community structure in maintaining soil function under legume-based rotation, providing guidance for agricultural management practice and global sustainable crop production.</jats:p>
          </jats:sec>