Afforestation is an effective approach to rehabilitate degraded ecosystems, but often depletes deep soil moisture. Presently, it is not known how an afforestation-induced decrease in moisture affects soil microbial community and functionality, hindering our ability to understand the sustainability of the rehabilitated ecosystems. To address this issue, we examined the impacts of 20 years of afforestation on soil bacterial community, co-occurrence pattern and functionalities along vertical profile (0-500 cm depth) in a semiarid region of China’s Loess Plateau. We showed that the effects of afforestation with a deep-rooted legume tree on cropland were greater in deep than that of in top layers, resulting in decreased bacterial beta diversity, more responsive bacterial taxa and functional groups, increased homogeneous selection, and decreased network robustness in deep soils (120-500 cm). Organic carbon and nitrogen decomposition rates and multifunctionality also significantly decreased by afforestation, and microbial carbon limitation significantly increased in deep soils. Moreover, changes in microbial community and functionality in deep layer was largely related to changes in soil moisture. Such negative impacts on deep soils should be fully considered for assessing afforestation’s eco-environment effects and for the sustainability of ecosystems because deep soils have important influence on forest ecosystems in semiarid and arid climates.