With abnormal global climate change and the frequent occurrence of extreme weather, low-temperature stress poses an increasingly serious threat to the diversity of plants. Low temperatures accompanied by limitations in nitrogen cause a series of morphological, physiological, and molecular changes in plants. Nitrogen dioxide (NO2) is a gas that is considered to be a toxic air pollutant. However, NO2 in the atmosphere can be absorbed by plants and participate in nitrogen metabolism. In this study, tobacco (Nicotiana tabacum L.) seedlings were fumigated with a concentration of 4 µL·L−1 NO2 at 4 ℃ for 10 days. NO2 promoted the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway in the leaves at low temperatures, which transforms more organic nitrogen that can be directly utilized by the plants, and improves the skeleton for carbon metabolism. Moreover, gamma-aminobutyric acid (GABA) was generated through the ornithine and glutamate pathways, and the biosynthesis of proline was also enhanced after treatment with NO2. Together, these compounds regulate the osmotic balance of tobacco leaf cells under low-temperature stress. NO2 activated the ascorbate-glutathione (AsA-GSH) cycle in the leaves under low-temperature stress, and this antioxidant enzyme system synergistically removed the intracellular free radicals that result from reactive oxygen species. Additionally, NO2 significantly increased the content of nitric oxide (NO) in the leaves under low-temperature stress and enhanced the opening of stomata, thus, improving photosynthesis in the leaves. The biosynthesis of chlorophyll in the leaves was inhibited by low-temperature stress, but NO2 promotes the biosynthesis of chlorophyll directly or through the nitric acid signaling pathway and improves the ability of plants to capture light energy. NO2 also alleviates the photoinhibition induced by cold stress by regulating photosynthetic electron transfer and absorbing more energy for the assimilation of photosynthetic carbon. This improves the photochemical efficiency of tobacco leaves. In conclusion, NO2 enhanced the tolerance of tobacco seedlings to low temperature by regulating nitrogen metabolism, the osmotic balance, antioxidant system, and photosynthesis.