This study tested the possible root biomass improvements in crop rotations after the conversion of grasslands, and crop samples from maize, winter wheat, and winter barley were collected during 2011-2013 from a long-term experimental site in Lusignan, France (http://www.soere-acbb). Root biomass C quantification was performed using delta C-13 isotopic signatures to determine the presence of both C3 and C4 plants. We also calculated the recovery rate of maize root biomass C. The results showed that after crop rotations, 0-60 cm root biomass C values were 44.1, 34.2, and 18.7 g C m(-2) for maize, winter wheat, and winter barley respectively. The Root biomass C of crops after conversion to grassland was approximately 2-3 times those observed after crop rotations. However, incorporating ley grassland duration into crop rotations showed limited improvements in shoot biomass C and grain yield of the crops, regardless of the decreased rate of N fertilizer for maize. Moreover, root biomass C had a significant relationship with N supply from residues (P<.05). Nevertheless, shoot biomass C of only maize showed significance in its relationships with N supply and root biomass C. In addition, in each 30 cm soil layer (0-30 cm, 30-60 cm, and 60-90 cm), the recovery rate of maize roots decreased to approximately 15% when root biomass C increased to 10 g C m(-2). However, further increases in root biomass C had little impact on the recovery rate. In conclusion, compared with continuous cropland, incorporating ley grassland duration into crop rotations increases root biomass C of crops, but this change may not be a significant increase of the shoot biomass C or grain yield. This finding simply indicates the improved C input from crops and the potential to increase soil organic C, as well as providing a model for the sustainability of crop rotations.