Fertilizer regime affects plant growth and soil microbial community composition, however, its impact on organic nitrogen (N) uptake by plants remains poorly understood. To address this, we undertook an in-situ, short-term uptake experiment based on 13C, 15N labelling, and 13C-PLFA analysis at two long-term (6 year) fertilizer trial sites (Jintan and Changshu). Each site had five treatments: a control without fertilizers, NPK fertilizers, 50% NPK fertilizer +6 t/ha pig manure, 100% NPK fertilizer + cereal straw, and 50% NPK fertilizer +6 t/ha pig manure and cereal straw. Overall, we found that 6–21% and 6–11% of the added 13C-15N-glycine was taken up intact by wheat, while 18–35% and 8–20% was captured by soil microorganisms in Jintan and Changshu locations, respectively. These results indicate that wheat has an appreciable capacity to utilize organic N, even in fertile agricultural soils. Organic N uptake by wheat correlated positively with ammonium and nitrate soil contents, indicating that inorganic N may enhance organic N capture by increasing plant biomass. The 13C:15N ratio in the microbial biomass showed that 32–71% and 13–71% of the 15N was absorbed through a direct uptake route in Jintan and Changshu soils. Chemical fertilizer reduced microbial biomass and increased the proportion of intact glycine uptake by wheat. Gram-positive bacteria accounted for 18–23%, and 13–15% of the total 13C labelled PLFA in Jintan and Changshu, respectively, while Gram-negative bacteria accounted for 43–48% and 66–72% indicating that they are the dominant competitors with plants for soil nutrients. Total 15N uptake by wheat and microorganisms was highest in the 50% NPK fertilizer + pig manure and cereal straw treatment at both sites, indicating that it represents the best fertilizer practice for sustainable food production, as it not only reduced chemical fertilizer application, improved wheat growth and microbial biomass, but also increased wheat utilization of soil organic N.