An unbiased partitioning of autotrophic and heterotrophic components of soil CO2 efflux is important to estimate forest carbon budgets and soil carbon sequestration. The contribution of autotrophic sources to soil CO2 efflux (FA) may be underestimated during the daytime as a result of internal transport of CO2 produced by root respiration through the transpiration stream. Here, we tested the hypothesis that carbon isotope composition of soil CO2 efflux (dFS) in a Eucalyptus plantation grown on a C4 soil is enriched during the daytime, which will indicate a decrease in FA during the periods of high transpiration. Mean dFS of soil CO2 efflux decreased to -25.7 parts per thousand during the night and increased to -24.7 parts per thousand between 11:00 and 15:00 h when the xylem sap flux density was at its maximum. Our results indicate a decrease in the contribution of root respiration to soil CO2 efflux during the day that may be interpreted as a departure of root-produced CO2 in the transpiration stream, leading to a 17 parts per thousand underestimation of autotrophic contribution to soil CO2 efflux on a daily timescale.