Conservation tillage and crop rotation diversification have been promoted to increase soil C and N storage; however, the interactive effects of tillage and crop rotation on soil C and N dynamics remain contradictory. Using a long-term (24-yr) experiment established at a clay loam site (Orthic Humic Gleysol) at Ridgetown, Ontario, Canada, the interactive effects of five crop rotations and two tillage systems were tested on soil organic C (SOC) and total N storage in soil depth increments and in the full soil profile (0-0.6 m) in 2019. While crop rotation influenced SOC and total N concentration in top 0.2 m, these effects were minimized when storage was expressed on an equivalent soil mass basis. Over the 0-0.6 m depth, no-tillage had 24 Mg SOC ha-1 and 4.7 Mg total N ha-1 greater content, respectively, than conventional tillage, supporting the value of no-tillage on increasing soil C and N in the long-term. Interestingly, no interactive effects of crop rotation and tillage on soil C and N storage in 0-0.6 m were observed. While the type of crop species and amount of C and N inputs under different crop rotations are important variables impacting the soil C and N storage, our results suggest that the crop rotation diversity was not a major driver of soil C and N in this study. Future mechanistic investigations exploring the persistence and linkage of soil C and N with crop rotation diversity in the tested production systems are needed.