Soil aggregates highly regulate nitrogen (N) turnover, yet their functions in regulating N retention under long-term crop rotation remain unclear. This study used ¹⁵N-labeled fertilizer N to investigate how different-sized soil aggregates regulate fertilizer N retention and redistribution under long-term crop rotation systems. The results revealed that large macroaggregates exhibited a more pronounced depletion of fertilizer N (enrichment factor, Ef: 0.73−0.95) than of total N (Ef: 0.93−1.00). In contrast, macroaggregates and microaggregates enriched fertilizer N (Ef: 1.00−1.16). Crucially, we found a temporal divergence: after harvest (with new ¹⁵N-labeled fertilizer application), macroaggregates preferentially sequestered new N (current-season fertilizer N), whereas microaggregates dominated the stabilization of old N (previous-year fertilizer N) in the absence of ¹⁵N-labeled fertilizer in 2024. This functional specialization is driven by aggregate turnover, whereby disintegrating macroaggregates release old N for stabilization in microaggregates, while reforming macroaggregates encapsulate new N. The accelerated release of fertilizer N from these macroaggregates during crop cultivation supplies N to crops, while their reformation concurrently enriches new N. This process underscores the dual functions of macroaggregates in both supplying N to crops and enriching new N. Ultimately, crop rotations reinforce this beneficial dynamic by restructuring soil aggregation, leading to a marked expansion of the soil N pool, with fertilizer N storage increasing by 27.11−111.68% and total N storage by 2.94−14.22% compared to continuous cropping. Our findings establish the functional heterogeneity of soil aggregates as a key mechanism for long-term fertilizer N retention and stabilization. This provides a mechanistic basis for optimizing N management under crop rotations.