Subsoils represent a large C reservoir in which carbon appears to be stabilized for centuries. We measured the mineralization rates of soil C in deep soil layers (80-100 cm) compared to the tilled layer (0-30cm) of a cultivated soil and investigated the factors responsible for the expected stabiliization of C in the long term. We hypothesed that limits to biodegradation in deep soil layers were (1) limiting N and P nutrients, (2) low O2 concentration , (3) physical protection of soil organic matter in aggregates or (4) unfavourable physiological state of microorganisms due to C depletion in the subsoil. We tested these hypotheses with a range of microscosms incubated in controlled conditions. Physical protection and the physiological state of microorganisms were the factors explaining slow biodegradation of organic matter in the subsoil.