The role of P availability on soil C sequestration is poorly understood in no‐tillage production. The objective of this study was to examine the long‐term phosphorus (P) fertilization effect on soil organic C (SOC), N, and C:N ratio in soil aggregates at different depths in no‐till corn (Zea mays L.)‐wheat (Triticum aestivum L.)‐soybean (Glycine max L.) rotations. The experiment was conducted on low to medium P soils at Springfield and Milan, TN during 2013–2015 from 0–15 cm and from 0–5, 5–10, 10–15, and 15–30 cm in 2018 using Latin square design with five replications of five P rates (0, 29, 59, 88, and 117 kg P ha−1) applied annually. Bulk soil and aggregate sizes > 2, 0.25–2, 0.053–0.25, and < 0.053 mm were analyzed for SOC and N. Phosphorus rate of 88 kg ha−1 resulted in high soil testing P and had higher SOC and N than all other treatments on low P soil. However, over‐application of P at 117 kg ha−1 decreased SOC on low P soil. No response of SOC or N to P fertilization was observed on medium P soil. Application of 29 kg P ha−1 increased large macroaggregate (> 2 mm) weight. Concentrations of SOC and N were higher in microaggregates (0.053–0.25 mm) than the other aggregates. In conclusion, SOC and N stocks are increased on low P soils with appropriate P application, which is crucial for simultaneous improvements of soil C sequestration and crop yield.