Plant communities around natural CO2 springs have been exposed to elevated CO2 levels over many generations and give us a unique opportunity to investigate the effects of long-term elevated CO2 levels on wild plants. We searched for natural CO2 springs in cool temperate climate regions in Japan and found three springs that were suitable for studying long-term responses of plants to elevated levels of CO2: Ryuzin-numa, Yuno-kawa and Nyuu. At these CO2 springs, the surrounding air was at high CO2 concentration with no toxic gas emissions throughout the growth season, and there was natural vegetation around the springs. At each site, high-CO2 (HC) and low-CO2 (LC) plots were established, and three dominant species at the shrub layers were used for physiological analyses. Although the microenvironments were different among the springs, dicotyledonous species growing at the HC plots tended to have more starch and less nitrogen per unit dry mass in the leaves than those growing at the LC plots. In contrast, monocotyledonous species growing in the HC and LC plots had similar starch and nitrogen concentrations. Photosynthetic rates at the mean growth CO2 concentration were higher in HC plants than LC plants, but photosynthetic rates at a common CO2 concentration were lower in HC plants. Efficiency of water and nitrogen use of leaves at growth CO2 concentration was greatly increased in HC plants. These results suggest that natural plants growing in elevated CO2 levels under cool temperate climate conditions have down-regulated their photosynthetic capacity but that they increased photosynthetic rates and resource use efficiencies due to the direct effect of elevated CO2 concentration.