Nitrogen fertilization can change soil respiration via microbial biomass, which affects the decomposition of soil organic matter (SOM) and plant residues. The quantitative influence and underlying mechanisms of mineral N and the combination of straw and N on stable organic N (humus) are still unclear. To explore the mechanisms of the conversion of mineral N to stable organic N (humus N), we performed incubation experiments using C-13 natural abundance and N-15 isotope labeling techniques with four treatments: control, added straw (St), added N (N), and added straw and N (St + N). The effect of N addition on the CO2 emission rate and cumulative CO2 emissions showed a prominent two-phase characteristic: both the CO2 emission rate and cumulative production were stimulated by exogenous mineral N during the initial stage, while the CO2 emission rate gradually decreased during the middle and later stages; however, residual C may remain in the soil for a longer time, leading to great differences in soil C/N ratios at different stages. This indicates that added N was rapidly utilized by microorganisms. At the end of the incubation period, the proportion of N-15 derived from labeled N to total N in humus was 0.063% (N) and 0.085% (St + N), while the proportion of N-15 in humus to total mineral N added was 0.41% and 0.64%, respectively. This indicated that even if only mineral N was added, the stable organic N in humus was converted by microbes. Compared with the addition of only mineral N, the combination of N and straw promoted the conversion of mineral N to stable organic N. The results of this study clarify the effect of mineral N on stable organic N and associated SOC mineralization, and can thus be used to develop strategies to improve soil fertility or mitigate climate change by increasing the SOC.