Tillage practices have a major effect on soil C storage and cropping sustainability, due to their impact on soil aggregation, organic residue decomposition rate, OC dynamics, microbial abundance and diversity, N mineralization and nutrient availability. Our research was aimed at assessing the long term effects of different tillage treatments on soil organic matter (SOM) quantity and quality and its evolution with time, in a loam textured-soil from central Italy cultivated with continuous maize. The tillage treatments included a conventional tillage (DP) by mouldboard ploughing to 40 cm depth, a ripper subsoiling (RS) to 40–45 cm, a shallow tillage by mouldboard ploughing to 20 cm depth (SP) and minimum tillage to 10 cm by disk harrowing (DH). The soil was sampled in 1999 and 2011 (after 5 and 17 years from the beginning of the trial, respectively), at depth increments of 0–10, 10–20, 20–30 and 30–40 cm and analysed for total organic C (TOC), OC recalcitrant and labile fractions by chemical hydrolysis, total N, bulk density, aggregate stability and hydraulic conductivity. After 17 year of treatments, the different tillage systems did not affect the overall amount of OC stored in a 0–40 cm equivalent soil mass; nevertheless, they produced significant differences in soil OC vertical distribution along the soil profile and OC recalcitrance. Both DH and RS increased soil TOC in the surface layer, with predominance of labile OC under DH and recalcitrant OC under RS. Differently, DP caused a net loss of recalcitrant OC, probably due to a detrimental impact on soil aggregate stability and, subsequently, on SOM physical protection. RS showed the largest potential for OC sequestration in stable form in the considered agroecosystem.