The priming effect is well acknowledged in soil systems but the effect of nitrogen (N) fertilization remains elusive. To explore how N modifies the priming effect in soil organic matter (SOM), one in situ experiment with ¹³C labeled glucose addition (0.4 mg C g^-1 soil, 3.4 atom % ¹³C) was conducted on soil plots fertilized with three gradients of urea (0, 4 and 16 g N m^-2 yr^-1). After glucose addition, the soil CO₂ concentration and phospholipid fatty acid (PLFA) were measured on day 3, 7, 21 and 35. The study found that N fertilization decreased soil CO₂, PLFA and the fungi to bacteria ratio. Glucose triggered the strongest positive priming in soil at 0 g N m^-2 yr^-2, meanwhile N fertilization decreased SOM-derived CO₂. Soil at 4 g N m^-2 yr^-2 released the largest amount of glucose-derived carbon (C), likely due to favorable nutrient stoichiometry between C and N. Stable microbial community biomass and composition during early sampling suggests “apparent priming” in this grassland. This study concludes that N fertilization inhibited soil priming in semi-arid grassland, and shifted microbial utilization of C substrate from SOM to added labile C. Diverse microbial functions might be playing a crucial role in soil priming and requires attention in future N fertilization studies.