Knowledge gaps regarding potential ontogeny and plant species identity effects on carbon isotope fractionation might lead to misinterpretations of carbon isotope composition (δ13C) of respired CO2, a widely-used integrator of environmental conditions. In monospecific mesocosms grown under controlled conditions, the δ13C of C pools and fluxes and leaf ecophysiological parameters of seven herbaceous species belonging to three functional groups (crops, forage grasses and legumes) were investigated at three ontogenetic stages of their vegetative cycle (young foliage, maximum growth rate, early senescence). Ontogeny-related changes in δ13C of leaf- and soil-respired CO2 and 13C/12C fractionation in respiration (ΔR) were species-dependent and up to 7‰, a magnitude similar to that commonly measured in response to environmental factors. At plant and soil levels, changes in δ13C of respired CO2 and ΔR with ontogeny were related to changes in plant physiological status, likely through ontogeny-driven changes in the C sink to source strength ratio in the aboveground plant compartment. Our data further showed that lower ΔR values (i.e. respired CO2 relatively less depleted in 13C) were observed with decreasing net assimilation. Our findings highlight the importance of accounting for ontogenetic stage and plant community composition in ecological studies using stable carbon isotopes.