Terrestrial vegetation currently absorbs approximately a third of the annual anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However,terrestrial net primary production is highly sensitive to atmospheric [CO2] and associated climatic changes. In C3-plants, which dominate terrestrial vegetation, netphotosynthesis depends on the ratio between gross photosynthesis and photorespiration, which strongly depends on [CO2]. However, our knowledge of feedbacks betweenterrestrial biomes and increasing atmospheric [CO2] is nearly entirely based on atmospheric inversion models and manipulation experiments, which do not reveal physiological mechanisms or are limited in duration and to step increases in [CO2]. By applying novel NMR (Nuclear Magnetic Resonance) spectroscopy methodology we examine isotopomer ratios of plant carbohydrates to probe shifts in the photosynthesis/photorespiration ratio in C3 plants over more than a century. Using herbarium samples of natural vascular plant species, crops and a Sphagnum species, we detect a consistent 35% increase in the 2photosynthesis/photorespiration ratio in responseto the ~100 ppm CO2 increase between approximately 1900 and 2013, with no evidencefor feedback regulation by the plants. Our data provide direct quantitative information on the “CO2 fertilization effect” over century time scales, thus addressing a major uncertainty in Earth system models, enabling improved predictions of the future [CO2] sink strength of terrestrial ecosystems. Further, relating the detected metabolic shift in crop plants to historic yield trends indicates that only a fraction of the increased net photosynthesis has translated into increased yield. Our results also demonstrate that archives of plant material contain metabolic information embedded in their isotopomer ratios covering centuries, bridging a fundamental gap between experimental plant science and paleoenvironmental studies.