Conservation agriculture (CA) has been reported to affect nutrient cycling. This study aims to investigate how CA induced soil organic matter stratification affects carbon and nitrogen turnover. A case farm study was established on two Danish farms with conventional ploughed tillage (P) and CA practises. Here, we studied how organic matter stratification patterns to 50 cm soil depth differed between the two systems. Further we investigated differences in carbon and nitrogen mineralization patterns in lab incubation experiments. Average stratification ratio, the ratio between soil C and N content in the upper 5 cm and at 20–30 cm, the depth of the plough layer in the ploughed system, was 1.86 and 1.61 under CA and 1.04 and 1.06 under P. Carbon respiration from intact soil core incubation was affected by soil total carbon content, and showed stronger stratification in CA than in P. Nitrogen mineralization rates from intact soil core incubation was largest in CA top-layer compared to CA 13.5–16.5 cm layer and both P soil layers, with initial mineralization followed by immobilization during the second half of the a four-week incubation. Net change in mineral nitrogen after incubation was only apparent in the 13.5–16.5 cm layer in P, with an average N mineralization rate of 0.08 mg N kg⁻¹ soil d⁻¹. Sieving to 2 mm did not affect N mineralization dynamics. Field-based ammonium to nitrate ratio was higher in CA than in P soils, across varying soil depths and time-points over the entire year. Soil acidity was reduced by one pH unit in CA compared to P. Microbial metabolic capacity was significantly larger in the top 5 cm of CA from the deeper depths, and from the P soils. In conclusion, carbon and nitrogen mineralization, as well as microbial metabolic capacity were strongly affected by an increased stratification ratio of organic matter in CA.