Hemicelluloses account for one-quarter of the global dry plant biomass and therefore are the second most abundant biomass fraction after cellulose. Despite their quantitative significance, the responsiveness of hemicelluloses to atmospheric carbon oversupply is still largely unknown, although hemicelluloses could serve as carbon sinks with increasing CO2 concentrations. This study aimed at clarifying the role hemicelluloses play as carbon sinks, analogous to non-structural carbohydrates (NSC), by experimentally manipulating the plants' carbon supply. Sixteen plant species from four different plant functional types (grasses, herbs, seedlings of broad-leaved trees and conifers) were grown for 2 months in greenhouses at either extremely low (140 ppm), medium (280 ppm) or high (560 ppm) atmospheric CO2 concentrations, thus inducing situations of massive C-limitation or -oversupply. Above and belowground biomass as well as NSC significantly increased in all species and tissues with increasing CO2 concentrations. Increasing CO2 concentrations had no significant effect on total hemicellulose concentrations in leaves and woody tissues in all species, except for two out of four grass species, where hemicellulose concentrations increased with atmospheric CO2 supply. Despite the overall stable total hemicellulose concentrations, the monosaccharide spectra of hemicelluloses showed a significant increase in glucose monomers in leaves of woody species as C-supply increased. In summary, total hemicellulose concentrations in de novo built biomass seem to be largely unaffected by changed atmospheric CO2 concentrations, while significant increases of hemicellulose-derived glucose with increasing CO2 concentrations in leaves of broad-leaved and conifer tree seedlings showed differential responses among the different hemicellulose classes in response to varying CO2 concentrations.