Climate and land-use changes modify plant rooting depth, signifying that organic matter with long residence times in deep soil layers can be exposed to rhizospheres and associated microbial activities. The presence of roots in soils stimulates mineralization of native soil C, via a process termed the rhizosphere priming effect (RPE), which may in consequence lead to loss of soil C. By growing a deep rooting grass, Festuca arundinacea, on soil columns and under continuous dual labelling (13C- & 14C-CO2), we show that root penetration up to 80 cm into a soil profile stimulated mineralization of ∼15,000 year-old soil C. The RPE, after normalization with root biomass, was similar along the soil profile indicating that deep C is as vulnerable to priming as surface C. The RPE was strongly correlated with respiration of plant-derived C, and a PLFA marker representative of saprophytic fungi (18:2ɷ6c) across all soil layers. Moreover, experimental disruption of soil structure further stimulated soil C mineralization. These findings suggest that the slow soil C mineralization in deep layers results from an impoverishment of energy-rich plant C for microorganisms (especially for saprophytic fungi), combined with a physical disconnection between soil C and microorganisms. Based on our results, we anticipate higher mineralization rates of deep millennia-old SOM in response to deeper root penetration which could be induced by changes in agricultural practices and climate.