Carbon isotope (¹³C) fractionations occurring during and after photosynthetic CO₂ fixation shape the carbon isotope composition (δ¹³C) of plant material and respired CO₂. However, responses of ¹³C fractionations to diel variation in starch metabolism in the leaf are not fully understood. Here we measured δ¹³C of organic matter (δ¹³C_OM), concentrations and δ¹³C of potential respiratory substrates, δ¹³C of dark-respired CO₂ (δ¹³C_R), and gas exchange in leaves of starch-deficient plastidial phosphoglucomutase (pgm) mutants and wild-type plants of four species (Arabidopsis thaliana, Mesembryanthemum crystallinum, Nicotiana sylvestris, and Pisum sativum). The strongest δ¹³C response to the pgm-induced starch deficiency was observed in N. sylvestris, with more negative δ¹³C_OM, δ¹³C_R, and δ¹³C values for assimilates (i.e. sugars and starch) and organic acids (i.e. malate and citrate) in pgm mutants than in wild-type plants during a diel cycle. The genotype differences in δ¹³C values could be largely explained by differences in leaf gas exchange. In contrast, the PGM-knockout effect on post-photosynthetic ¹³C fractionations via the plastidic fructose-1,6-bisphosphate aldolase reaction or during respiration was small. Taken together, our results show that the δ¹³C variations in starch-deficient mutants are primarily explained by photosynthetic ¹³C fractionations and that the combination of knockout mutants and isotope analyses allows additional insights into plant metabolism.