Anthropogenic activities have raised nitrogen (N) input worldwide with profound implications for soil carbon (C) cycling in ecosystems. The specific impacts of N input on soil organic matter (SOM) pools differing in microbial availability remain debatable. For the first time, we used a much‐improved approach by effectively combining the ¹³C natural abundance in SOM with 21 years of C₃–C₄ vegetation conversion and long‐term incubation. This allows to distinguish the impact of N input on SOM pools with various turnover times. We found that N input reduced the mineralization of all SOM pools, with labile pools having greater sensitivity to N than stable ones. The suppression in SOM mineralization was notably higher in the very labile pool (18%–52%) than the labile and stable (11%–47%) and the very stable pool (3%–21%) compared to that in the unfertilized control soil. The very labile C pool made a strong contribution (up to 60%) to total CO₂ release and also contributed to 74%–96% of suppressed CO₂ with N input. This suppression of SOM mineralization by N was initially attributed to the decreased microbial biomass and soil functions. Over the long‐term, the shift in bacterial community toward Proteobacteria and reduction in functional genes for labile C degradation were the primary drivers. In conclusion, the higher the availability of the SOM pools, the stronger the suppression of their mineralization by N input. Labile SOM pools are highly sensitive to N availability and may hold a greater potential for C sequestration under N input at global scale.