The hydrogen isotopic composition (δ2H) of plant compounds is increasingly used as a hydro-climatic proxy, however, the interpretation of δ2H values is hampered by potential co-effecting biochemical and biophysical processes. Here, we studied δ2H values of water and carbohydrates in leaves and roots, and of leaf n-alkanes, in two distinct tobacco (Nicotiana sylvestris) experiments. Large differences in plant performance and biochemistry resulted from (a) soil fertilization with varying nitrogen (N) species ratios and (b) knockout-induced starch deficiency. We observed a strong 2H-enrichment in sugars and starch with a decreasing performance induced by increasing NO3-/NH4+ ratios and starch deficiency, and from leaves to roots. However, δ2H values of cellulose and n-alkanes were less affected. We show that relative concentrations of sugars and starch, interlinked with leaf gas-exchange, shape δ2H values of carbohydrates. We thus provide novel insights into drivers of hydrogen isotopic composition of plant compounds and the mechanistic modelling of plant cellulose δ2H values.