Long-term effect of biochar on soil C sequestration of recent carbon inputs has been examined using soil from arable fields in Belgium with charcoal-enriched black spots dating >150 years ago from historical charcoal production mound kilns. Topsoils from these 'black spots' had a higher organic C concentration ± 0.9% organic carbon (OC) than adjacent soils outside these black spots (2.1 ± 0.2% OC). The soils had been cropped with maize for at least 12 years which provided a continuous input of C with a C isotope signature (δ13C) −13.1, distinct from the δ13C of soil organic carbon (−27.4 ‰) and charcoal (−25.7 ‰) collected in the surrounding area. The isotope signatures in the soil revealed that maize-derived C concentration was significantly higher in charcoal-amended samples ('black spots') than in adjacent unamended ones (0.44% vs. 0.31%; P = 0.02). Topsoils were subsequently collected as a gradient across two 'black spots' along with corresponding adjacent soils outside these black spots and soil respiration, and physical soil fractionation was conducted. Total soil respiration (130 days) was unaffected by charcoal, but the maize-derived C respiration per unit maize-derived OC in soil significantly decreased about half (P < 0.02) with increasing charcoal-derived C in soil. Maize-derived C was proportionally present more in protected soil aggregates in the presence of charcoal. The lower specific mineralization and increased C sequestration of recent C with charcoal are attributed to a combination of physical protection, C saturation of microbial communities and, potentially, slightly higher annual primary production. Overall, this study provides evidence of the capacity of biochar to enhance C sequestration in soils through reduced C turnover on the long term. (Hernandez-Soriano "et al", 2015).