•Conservation agriculture management system affected the SOC depth distribution.•C accumulation was observed in conservation agriculture only in the first 30cm.•C accumulation was affected by mineral clay composition.•Conservation management led to higher polycondensed humic molecules content.•Soil tillage management did not affect microbial C and N contents. Conservation agriculture is one of the agro-environment measures promoted by the Veneto Region (North-eastern Italy) to regulate and support many ecosystem services. This study compared conventional and conservation agriculture management systems in order to evaluate their effects on both SOC stocks and quality i.e. humic C and its molecular weight fractions, microbial C and N. The experiment was set up in 2010 on three farms in Veneto Region. In order to improve the monitoring procedures, a massive soil sampling programme was conducted in 2011 and 2014 in ca. 150 positions, considering the SOC stratification within a 0–50cm profile. Results suggested that conservation agriculture practices affected SOC distribution rather than its total amount. The retention of crop residues on the soil surface and the absence of tillage operations drove SOC dynamics in the top layer (0–5cm) of the conservation system, while residues incorporation with ploughing was responsible for SOC accumulation at the 30–50cm depth in the conventional one. SOC stock variation in the conservation treatment was also influenced by root C input, which was identified as a major factor able to promote SOC accumulation in the 0–30cm profile. The role of clay on SOC dynamics was not uniform in the three farms since it depended both on the clay amount and its mineral composition. The strong interactions existing between management systems and local soil conditions were also confirmed by the C quality analyses. This research did not demonstrate the benefits of conservation practices on SOC sequestration during the transition period. However, SOC sequestration is only one of the numerous ecosystem services provided by conservation practices. Some of these depend on the C content and quality in the top layers that, as demonstrated in our work, were strongly affected by the C stratification processes triggered by conservation agriculture.