Plant leaves absorb atmospheric nitrogen dioxide (NO2) primarily via the stomata. Studies of changes in plant growth and physiology after exposure to NO2 are limited. Therefore, this study investigated the physiological response of Carpinus betulus (european hornbeam) chloroplasts after NO2 exposure using fumigation equipment that was able to control timing and record NO2 concentrations. The NO2 concentration was 6 µL·L−1. Seven treatment durations (0, 1, 6, 12, 24, 48, and 72 hours) were designed. After fumigation, plants recovered for 30 days under greenhouse conditions. The physiological response, stomatal behavior, thicknesses of palisade and spongy tissues, and chloroplast ultrastructure were measured. In the 48-hour and 72-hour NO2 treatment groups, the chloroplast contents and net photosynthesis rates of the leaves decreased, palisade and spongy tissues thickened, and chloroplast thylakoids swelled; however, the 1-hour NO2 treatment did not have a noticeable toxic effect on C. betulus leaves. After 30 days of recovery, the plants returned to their natural growth level by increasing the chloroplast content and enhancing net photosynthesis. Short durations and high concentrations of NO2 exposure had significantly negative impacts on the physiological response of C. betulus; however, this toxic effect of high NO2 concentrations on C. betulus can be recovered by restoration of unpolluted air. The results of this study may provide a scientific reference and an additional choice of plants species for the application of C. betulus in functional gardening design and ecological green space construction.