Knowledge gaps regarding potential ontogeny and plant species identity effects on carbon isotope fractionation might lead to misinterpretations of carbon isotope composition (delta C-13) of respired CO2, a widely-used integrator of environmental conditions. In monospecific mesocosms grown under controlled conditions, the delta C-13 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 delta C-13 of leaf-and soil-respired CO2 and C-13/C-12 fractionation in respiration (Delta(R)) were species-dependent and up to 7 parts per thousand, a magnitude similar to that commonly measured in response to environmental factors. At plant and soil levels, changes in delta C-13 of respired CO2 and Delta(R) 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 above-ground plant compartment. Our data further showed that lower Delta(R) values (i.e. respired CO2 relatively less depleted in C-13) 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.