Soil microbial nitrate (NO3-) immobilization plays a vital role in enhancing the nitrogen (N) retention in the subtropical montane agricultural landscapes. However, how and why the potential microbial NO3- immobilization and the relative contribution of fungi and bacteria vary across different land use types remain still unclear in the subtropical mosaic montane agricultural landscapes. Thus, in the present study, soil gross microbial NO3- immobilization rates as well as the respective contribution of fungi and bacteria were determined throughout the whole soil profiles for three land use types (woodland, orchard, and cropland) by using the N-15 tracing and amino sugar-based stable isotope probing (Amino sugars-SIP) techniques. The soil gross microbial NO3- immobilization rates in woodland soils were significantly higher than those in cropland and orchard soils across different soil layers (p < 0.05), and those of topsoil were significantly higher than those for subsoils (e.g., 20-40 cm) across different land use types (p < 0.05). Soil microbial biomass C (MBC) and N (MBN), organic C (SOC), total N (TN), and dissolved organic C (DOC) contents and C/N ratios were closely associated to gross microbial NO3- immobilization rates. Fungi played a greater role than bacteria in immobilizing soil NO3- in woodland and orchard soils, but the opposite occurred in cropland soils that over 85% of the variations in fungal and bacterial NO3- immobilization rates could be explained by their respective phospholipid fatty acid-derived (PLFA-derived) biomass. The present study indicated that afforestation may be effective to enhance soil NO3- retention in alkaline soils, thereby likely decreasing the risk of NO3- losses in subtropical mosaic montane agricultural landscapes through enhancing the soil NO3- immobilization by both fungi and bacteria.