The conversion of grasslands to croplands is common in the agro-pastoral ecotone and brings potential risks to soil health and environmental safety. As the forming unit of soil structure, the status of soil aggregates determines soil health and is affected by multiple factors. This study investigated the changes in soil aggregate and main related factors in conversion grasslands with different managed years. Grassland conversion ages were selected as experimental treatments, which included unmanaged grassland, 3 years, 10 years, 30 years, and 50 years since grassland conversion. After grassland conversion, the proportion of large macro-aggregates with a particle size of >2 mm in the 0–10 cm soil layer decreased, small macro-aggregates with a particle size of 2–0.25 mm and micro-aggregates with a particle size of 0.25–0.053 mm increased, while aggregates with a particle size of <0.053 mm had no significant change. Soil chemical properties, most microorganisms and the soil aggregate stability indices MWD and GMD decreased at the early stage (<30 years) of the managed grasslands. After about 50 years of cultivation, soil chemical properties and microorganisms returned to equal or higher levels compared to unmanaged grasslands. However, the stability of aggregates (mean weight diameter (MWD) and geometric mean diameter (GMD)) did not recover to the initial state. MWD and GMD were positively correlated with most bacterial factors (total phospholipid fatty acids (PLFAs), bacteria, Gram-positive bacteria, Gram-negative bacteria, actinomycetes and arbuscular mycorrhizal fungi (AMF)) and some soil chemical properties (carbon, nitrogen and polysaccharides). According to the partial least square structural equation model, soil organic carbon, total nitrogen and phosphorus in the 0–10 cm soil layer explained 33.0% of the variance in MWD by influencing microorganisms. These results indicated that the stability of aggregates was directly driven by microorganisms and indirectly affected by soil organic carbon, total nitrogen and phosphorus.