Nitrogen dioxide (NO2) is a major air pollutant that affects plant growth, development and yields. Previous studies have found that atmospheric NO2 changes plant photosynthesis in a concentration-dependent manner. Low concentrations of NO2 (4.0 μL L−1) can increase photosynthetic rates, while high concentrations of NO2 (16.0 μL L−1) can have an inhibitory effect. However, the specific effects of a critical intermediate concentration of NO2 on the photosynthetic apparatus of plants has remained unknown. Therefore, in this study, tobacco seedlings at three-leaf ages were fumigated with a intermediate concentration of 8.0 μL L−1 NO2 for 15 days to determine the effects on leaf weight, leaf number per plant, chlorophyll content, net photosynthetic rate, the reaction center activity of photosystems I and II (PSI and PSII, respectively) and core protein gene expression (PsbA and PsaA). Fumigation with 8.0 μL L−1 NO2 increased the number of leaves per plant and the weight of leaves, and the leaves became dark green and curly after 10 days of fumigation. During NO2 fumigation for 15 days, the chlorophyll content, PSII maximum photochemical efficiency (Fv/Fm), electron transfer rate (ETR) and non-photochemical quenching (NPQ) increased most in the oldest leaves (Lmax leaves), but decreased PSI activity (∆I/Io). The Fv/Fm, ETR and NPQ in the youngest leaves (Lmin leaves) were lower than those of Lmax leaves, but the actual photochemical efficiency (ΦPSII) of PSII increased most and ∆I/Io was the highest in these samples. The Fv/Fm, ETR, NPQ and ΦPSII in the leaves at the middle leaf age (Lmid leaves) were lower than those of Lmin and Lmax leaves, but the relative fluorescence intensity of point L (VL) and the relative fluorescence intensity of point K (VK) decreased the most in these samples. Thus, this critical concentration of atmospheric NO2 increased the activity of PSII and inhibited PSI activity in expanded leaves of tobacco seedlings.