Background and Aims: Tillage system and crop rotation influences soil organic carbon (SOC)and total N (TN), but there remains considerable uncertainty in the response of C and N dynamics to fertilizer N inputs. Along-term (11-yr) experiment on a clay loam Orthic Humic Gleysol at Ridgetown, Ontario, Canada was used to evaluate the impact of fertilizer N applications(in-season zero N (-N) compared to (+N) 100 and 80 kg N ha-1yr-1to corn (Zea mays L.) and wheat(Triticum aestivum L.), respectively) on soil attributes. Methods: The cropping systems consisted of continuous corn (CC), corn-soybean(Glycine max L.)(C-S) and corn-soybean-wheat (C-S-W)rotations using conventional moldboard plough and no-till systems.Soil was collected from the 0-120 cm profile in 5, 10, and 20 cm increments and analyzed for SOC and TN. Results: The effect and interaction of N fertilization on soil attributes was highly dependent on crop rotation and tillage system.The gain in SOC and TN contents due to +N fertilizer was greatest (up to 31 and 57% relative to the -N control, respectively) in the 0-20 cm depth with the C-S-W rotation, and lowest under CC, which showed no N fertilizer effect.However, differences in SOC and TN were not confined to the surface 20 cm, as N fertilizer treatments significantly influenced the contentsat20-60 and 60-120 cm in certain rotation and tillage systems;C-S-W was the most responsive to N fertilizer-induced SOC and TN gains.Conclusions: Using regression analysis, we found that higher SOC contents corresponded to lower variability in the 5-yr mean corn yield, which suggests that the inclusion of winter wheat in a C-S rotation may have important implications for sustainable and resilient agroecosystems in humid, temperate climates.