Microbial biomass plays a critical role in soil organic carbon (SOC) mineralization. However, the effects of microbial biomass size on SOC mineralization are poorly understood. We investigated how the priming effect (PE) of plant residue inputs on native SOC mineralization responds to changes in microbial biomass size and nitrogen (N) availability in the same soil with a 23 y history of crops and grass cover with contrasting SOC contents. The size of the soil microbial biomass was changed by pre-incubating soils with glucose. The pre incubated soils were then treated with C-13-labeled ryegrass residue combined with or without N to determine the PE. In all soils, the addition of ryegrass residue significantly increased cumulative carbon dioxide (CO2) production, whereas the addition of N decreased it compared to the control (no ryegrass or N addition). After a 42 day incubation, only 9-16% of the ryegrass carbon (C) was mineralized to CO2, which contributed approximately 55 and 34% to the total CO2 production in crop and grass soils, respectively. The addition of N decreased CO2 production during the ryegrass decomposition by 9-45%, while the change in soil microbial biomass size had no impact. In addition, a positive PE was generally found in the soils amended with ryegrass alone, while the application of ryegrass residue combined with N decreased the PE. Moreover, the priming effect was independent of the size of the microbial biomass in crop soil. However, we observed a significant interaction of microbial biomass size and N availability on the priming effect in a grass soil but not in a crop soil. Our results indicate that soil microbial biomass size and soil C influence the magnitude and direction of the priming effect from C inputs depending on N availability.