Disentangling the autotrophic and heterotrophic components of soil CO2 efflux is critical to understanding the role of soil system in terrestrial carbon (C) cycling. In this study, we combined a stable C-isotope natural abundance approach with the trenched plot method to determine if root exclusion significantly affected the isotopic composition (δ13C) of soil CO2 efflux (RS). This study was performed in different forest ecosystems: a tropical rainforest and two temperate broadleaved forests, where trenched plots had previously been installed. At each site, RS and its δ13C (δ13CRs) tended to be lower in trenched plots than in control plots. Contrary to RS, δ13CRs differences were not significant. This observation is consistent with the small differences in δ13C measured on organic matter from root, litter and soil. The lack of an effect on δ13CRs by root exclusion could be from the small difference in δ13C between autotrophic and heterotrophic soil respirations, but further investigations are needed because of potential artefacts associated with the root exclusion technique.