Exogenous carbon (C) input may induce priming effects, leading to the loss of soil organic C (SOC) by accelerating the decomposition of native soil organic matter (SOM), while also replenishing SOC through various mechanisms. However, the net C balance resulting from priming and replenishment of SOC under long-term nitrogen (N) fertilization and its stoichiometric regulation mechanisms remain largely undetermined. Soils subjected to 11 years of different N applications were used to investigate the net C balance following the addition of exogenous 13C-labeled glucose. The retention of glucose-derived C exceeded the loss of C caused by the priming effect, resulting in a positive net C balance, albeit attenuated by historical N application (ranging from 25.9 to 36.9 μg C mg−1 SOC). The application of increasing historical N levels resulted in a decrease in soil C:N imbalance and an increase in soil N:phosphorus (P) imbalance, as well as an increase in TERC:N and TERC:P. This suggested that the C and/or P limitations of soil microbial communities were intensified with increased N availability. Soil nutrient stoichiometric imbalance and available resource stoichiometry directly influenced the threshold element ratio, which in turn impacted glucose mineralization, subsequently affecting the net C balance. Collectively, our results provided solid evidence that labile C input could lead to a positive net C balance, which diminished with increased historical N application and was primarily regulated by soil C:N:P stoichiometry. This study highlights the significant implications for the soil C turnover and sequestration under long-term N application management in agroecosystems.