•N fertilization and tillage reversal increased organic C more for soil with low C.•Macroaggregates were the most important fraction for C storage.•N fertilization and tillage reversal enhanced macroaggregate formation in topsoils.•Tillage reversal did not offset N fertilization-increased C storage in topsoils. Nitrogen (N) fertilization and reversing land management from long-term no tillage (NT) to conventional tillage (tillage reversal; TR) may markedly alter soil carbon (C) dynamics. We studied the impact of N fertilization, N applied at 100 kg ha−1 yr−1 (N100) vs. no N application (N0), and tillage reversal (NT vs. TR) on soil aggregation and aggregate-associated C in top- (0–10) and subsoils (50–60 cm) in a Black Chernozem and a Gray Luvisol. Our results showed that soil organic C content was greater in N100 (1.74 g C kg−1) than in N0 (1.29 g C kg−1), and in TR (1.71 g C kg−1) than in NT (1.32 g C kg−1) only in the topsoil of the Gray Luvisol. Nitrogen fertilization and tillage reversal improved topsoil, but not subsoil mean weight diameter or the amount of large macroaggregates (>2000 μm). Topsoil aggregate-associated C was only increased (P < 0.01) with N fertilization. Microaggregate-associated C in the topsoil of the Black Chernozem was 70.6 g C kg−1 sand-free water-stable aggregate in N100, which was 13.8% higher (P < 0.01) than that in N0. However, C associated with all aggregate fractions in the topsoil was increased (P < 0.05) by N fertilization in the Gray Luvisol. In the Black Chernozem, the physical protection for C in the subsoil was decreased by N fertilization and tillage reversal, as indicated by decreased large macroaggregate and microaggregate-associated C (P = 0.04 and < 0.001, respectively). Subsoil aggregate-associated C was not influenced by N fertilization or tillage reversal in the Gray Luvisol. We conclude that N fertilization and/or tillage reversal improved topsoil aggregation; only N fertilization improved the physical protection for C in the topsoil. The adoption of tillage reversal (2 years) did not offset the benefit of N fertilization on soil aggregation and soil C concentration.