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Uncertainty about long-term leaf-level responses to atmospheric CO2 rise is a major knowledge gap that exists because of limited empirical data. Thus, it remains unclear how responses of leaf gas exchange to elevated CO2 (eCO(2)) vary among plant species and functional groups, or across different levels of nutrient supply, and whether they persist over time for long-lived perennials. Here, we report the effects of eCO(2) on rates of net photosynthesis and stomatal conductance in 14 perennial grassland species from four functional groups over two decades in a Minnesota Free-Air CO2 Enrichment experiment, BioCON. Monocultures of species belonging to C-3 grasses, C-4 grasses, forbs, and legumes were exposed to two levels of CO2 and nitrogen supply in factorial combinations over 21 years. eCO(2) increased photosynthesis by 12.9% on average in C-3 species, substantially less than model predictions of instantaneous responses based on physiological theory and results of other studies, even those spanning multiple years. Acclimation of photosynthesis to eCO(2) was observed beginning in the first year and did not strengthen through time. Yet, contrary to expectations, the response of photosynthesis to eCO(2) was not enhanced by increased nitrogen supply. Differences in responses among herbaceous plant functional groups were modest, with legumes responding the most and C-4 grasses the least as expected, but did not further diverge over time. Leaf-level water-use efficiency increased by 50% under eCO(2) primarily because of reduced stomatal conductance. Our results imply that enhanced nitrogen supply will not necessarily diminish photosynthetic acclimation to eCO(2) in nitrogen-limited systems, and that significant and consistent declines in stomatal conductance and increases in water-use efficiency under eCO(2) may allow plants to better withstand drought.