Aims Understanding the patterns and drivers of carbon isotope discrimination ((13)Delta) in C-3 and C-4 functional groups is critical for predicting C-3/C-4 vegetation ratio from the isotopic composition of soil organic matter. In this study, we aimed to evaluate how intraspecific variation will modify functional group-level (13)Delta values and the associated prediction of C-3/C-4 vegetation ratio. Methods We investigated (13)Delta of 726 individual plants (96 species; C-3 and C-4 functional groups) and topsoil organic matter in 26 grassland communities along an aridity gradient in northern China. The fraction of C-4 contribution was calculated with mixing models that considered: (i) both intra- and interspecific effects on the (13)Delta values of C-3 and C-4 functional groups; (ii) only interspecific effects; or (iii) none of these effects. Important Findings We found divergent responses of plant (13)Delta at the intraspecific level to the changes of aridity across the gradient. The (13)Delta of both C-3 and C-4 functional groups was negatively correlated with an aridity index, with higher sensitivity for C-3 than for C-4 functional groups. Intraspecific (13)Delta variation played a key role in driving the total (13)Delta variations of C-3 plants. Overlooking such intraspecific effect in mixing models led to a greatly increased fraction of C-4 contribution to soil organic carbon. A correction for the effects of intraspecific variation is therefore essential for correctly inferring C-3/C-4 vegetation ratio in the past. Our findings provide basic information for the reconstruction of past vegetation change from bulk materials in arid and semiarid biomes.