AbstractNitrogen (N) released from soil organic matter (SOM) is quantitatively important for crop uptake, even when adequate fertiliser N is supplied. Understanding of SOM has shifted to recognise distinct fractions that correlate with properties such as turnover time, carbon (C) and N content, and chemical composition. Yet, how these fractions relate to N supply from SOM is poorly understood. This study aimed to link N mobilisation and C stability in coarse (≥50 μm) and fine (≤50 μm) fractions, and evaluate the roles of these fractions in supplying N in cropping soil. Soils from long‐term continuous cotton, cotton‐vetch, and cotton‐wheat rotations and a nearby uncleared site (NV) were separated into coarse and fine fractions, left as whole soil, or dispersed and freeze‐dried as a fractionation control. Initial C chemistry in fractions and whole soils was measured by solid state ¹³C NMR spectroscopy. N mobilisation and CO₂ loss were measured over a 14‐day incubation experiment. In the cropping soils, net immobilisation of N was measured in the separate fractions, while net mobilisation was found in the whole soils. In the NV soil, N mobilisation was greater in the fine fraction. C mineralisation followed the order fine fraction > fractionation control = coarse fraction > whole soil. C stability was best explained by physical protection within whole soil structure rather than chemical recalcitrance or mineral stabilisation. The results revealed an unexpected contrast between C and N mineralisation from SOM fractions and demonstrated the importance of soil aggregates for SOM stability. We show a cautionary impact of fractionation on C and N dynamic, highlighting the need for further research to understand the synergistic behaviour of SOM fractions in whole soils.