Environmental factors and physiological controls on photosynthesis influence the carbon isotopic signature of ecosystem respiration. Many ecosystem studies have used stable carbon isotopes to investigate environmental controls on plant carbon transfer from above- to belowground. However, a clear understanding of the internal mechanisms underlying time-lagged responses of carbon isotopic signatures in ecosystem respiration to environmental changes is still lacking. This study addressed plant physiological controls on the transfer time of recently assimilated carbon from assimilation to respiration. We produced a set of six wheat plants with varying physiological characteristics, by growing them under a wide range of nitrogen supply and soil water content levels under standardised conditions. The plants were pulse-labelled with 13C-CO2, and the isotopic signature of CO2 respired in the dark by plants and soil was monitored continuously over two days. Stomatal conductance (gs) was strongly related to the rate of transfer of recently assimilated carbon belowground. The higher gs, the faster newly assimilated carbon was allocated belowground and the faster it was respired in the soil. Our results suggest that carbon sink strength of plant tissues may be a major driver of transfer velocity of recently assimilated carbon to plant respiratory tissues and soil respiration.