Biomass as a renewable energy source has become increasingly prevalent in Europe to comply with greenhouse gas emission targets. As one of the most efficient perennial bioenergy crops, there is great potential in the Upper Rhine Region to explore biomass utilization of Miscanthus to confront climate change and land use demand in the future. Yet, the impacts of Miscanthus cultivation on soil quality have not been adequately explored. This study investigated the soil profiles of five- and 20-year-old Miscanthus fields (1 m depth) as well as grassland for reference in eastern France and Switzerland. The soil organic carbon (SOC) concentrations and δ13C compositions of four soil layers (0-10 cm, 10-40 cm, 40-70 cm and 70-100 cm) were determined. The CO2 emission rates of the topsoil were monitored for 42 days. Our results showed that Miscanthus, in general, could increase the SOC stocks compared to grassland, but the benefits of SOC sequestration were constrained to the surface soil. Isotopically, the Miscanthus-derived SOC ranged from 69% in the top 10 cm of soil down to only 7% in the 70 cm to 100 cm layer. This result raises the risk of overestimating the total net benefits of Miscanthus cultivation, when simply using the greater SOC stocks near the surface soil to represent the SOC-depleted deep soil layers. The Miscanthus fields had greater CO2 emissions, implying that the Miscanthus fields generated greater ecosystem respiration, rather than larger net ecosystem exchanges. Compared to the grassland soils, the surface soils of the Miscanthus fields tended to have a risk of acidification while having higher concentrations of phosphorus and potassium, calling for the inclusion of soil characteristics and SOC stability when evaluating the impacts of long-term Miscanthus cultivation on both current and future land use changes.