The effect of grassland conversion to cropland on the incorporation of N into microbial residues remains unclear. We analyzed for amino sugars, as a microbial residue biomarker, and adopted stable-isotope-probing technique to trace the assimilation of 15NH4+-N (added in the form of 15 N-labelled ammonium sulphate) into amino sugars with or without organic C sources (glucose and crop residue) in native grassland and converted cropland soils over a 65-day period. Total 15 N-amino sugars in the cropland were significantly decreased by 16–29% compared to grassland regardless of C source. This suggests that the microbial assimilation of available N into microbial residues was decreased, after conversion, and that stronger responses to substrate inputs occurred in the grassland soils. The addition of available and complex C substrates triggered divergent accumulation patterns of 15 N-amino sugars, indicating that C availability was a major driver for microbial N immobilization. Meanwhile, the conversion of grassland led to suppressed N immobilization activity of both fungi and bacteria. These results suggest that long-term land-use change could considerably affect the N utilization by fungi and bacteria during their anabolic processes, which may exert long-lasting impacts on soil organic N storage.