Soil microbial diversity affects soil chemical cycling and ecosystem function. However, how highly connected keystone species regulate associated microbial structure and function (of the whole community and of specialized species), irrespective of their abundance under land use change, remains unresolved. Here, we identified the relationship between microbial diversity [soil phospholipid fatty acid (PLFA) richness and composition, and ammonia-oxidizing archaea and bacteria (AOA and AOB) communities] with both a broad (i.e., microbial respiration) and specialized function (i.e., nitrification rate) in a woodland, shrubland, and adjacent cropland in subtropical China. Microbial respiration was significantly positively related to the microbial richness, but negatively correlated with the keystone species richness across different land use types. The relationships between biodiversity and the broad ecosystem function varied with land use, with a stronger relationship in the afforested land compared with in the cropland. In contrast to the broad function, land use did not significantly affect the specialized function, but the specialized function was positively related to AOA richness in the cropland. Additionally, the specialized function was predominately driven by the keystone species composition in AOA and AOB communities and indirectly regulated by soil environmental factors (particularly soil temperature) across land uses. Overall, our results provide direct experimental insight into the mechanisms underlying the role of the keystone species in regulating belowground ecosystem functions under land use change. In particular, our findings reveal that the broad function is maintained by the niche compensation effect, while the specialized function is maintained by the identity effect.