Abstract: To explore the response mechanisms of different low-temperature tolerant tobacco varieties to low-temperature stress, two tobacco cultivars, the cold-tolerant Tianxinyan and the cold-sensitive H382, were used as experimental materials in this study. The seedlings were cultivated to the seven-leaf-one-heart stage via substrate pot culture, followed by the setup of gradient recovery temperature treatments after low-temperature stress. The variation patterns of seedling biomass, photosynthetic characteristics, chlorophyll fluorescence characteristics and antioxidant metabolism indices were determined and analyzed. The results showed that low-temperature stress significantly inhibited leaf area expansion and dry matter accumulation of the two cultivars, and simultaneously reduced photosynthetic parameters(A, G_s, E and C_i). The chlorophyll fluorescence parameters(F_o, F_m and F_v) of both cultivars maintained high levels under suitable temperatures. After low-temperature and rewarming treatments, F_o of H382 increased significantly, while F_m and F_v decreased sharply, with the degree of PS Ⅱ functional damage being significantly higher in H382 than in Tianxinyan. Low-temperature stress induced the increases in the contents of malondialdehyde(MDA) and hydrogen peroxide(H₂O₂), as well as the activities of antioxidant enzymes such as SOD, POD and CAT in tobacco seedlings. Notably, Tianxinyan exhibited a more rapid response of antioxidant enzymes, and its reactive oxygen species scavenging efficiency and the recovery capacity of physiological indices after temperature recovery were significantly superior to those of H382. In conclusion, the cold-tolerant cultivar Tianxinyan can enhance its low-temperature tolerance by maintaining the stability of the photosynthetic system and activating the synergistic regulatory network of antioxidant enzymes.