Tobacco is an important economical leaf crop and complex model organism that is cultivated worldwide. Low temperature is the one of major factors causing abiotic damage to flue-cured seedlings; this damage can affect the growth and development of flue-cured tobacco seedlings and can decrease the yield and quality of flue-cured tobacco leaves. Currently, technologies for cultivating seedlings by using water and oxygen have been widely adopted in southern tobacco-cultivating regions. However, temperature is dependent on climate, which is difficult to control artificially. Low temperatures during the early spring season limit the culture of strong seedlings in southern tobacco-cultivation areas. The objective of this study was to analyze physiological and ecological adaptations of flue-cured tobacco seedlings by measuring parameters related to levels of photosynthetic, oxidant, and antioxidant factors in the cell membranes of leaves. We also used digital gene expression profiling technology to analyze the differential expression of genes in flue-cured tobacco seedlings after chilling stress. Seedlings of flue-cured tobacco strain K326 were used as experiment materials. Tobacco seedlings with 5-6 true leaves were divided in two groups. One group of flue-cured seedlings was placed in a light incubator at a low temperature range of 5 ℃ (night) to 7 ℃ (day) for 3 days as chilling stress treatment. Seedlings in the other (control) group remained at the initial temperature and illumination conditions and grew at a normal temperature range of 23 ℃ (night) to 25 ℃ (day), with other similar conditions. After chilling stress for 3 days, total chlorophyll (Chl), chlorophyll a (Chl a), and chlorophyll b (Chl b) contents decreased significantly by 2.88%-6.82%, and the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration (Tr) decreased significantly by 14.14%-68.50% compared with that of seedlings grown under the favorable temperature. Intercellular CO₂ concentration (Ci) showed no obvious changes. In terms of the membrane oxidant levels of leaves from flue-cured tobacco seedlings, proline and malondialdehyde (MDA) contents and electrolyte permeability increased significantly by 3.88%-144.22% compared with that of control seedlings, while the oxyradical generation rate decreased significantly by 10.66%. Additionally, the membrane antioxidant capacity of flue-cured tobacco seedlings and the activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly by 6.07% and 45.64%, respectively, compared with that grown under the favorable temperature, while peroxidase (POD) activity was significantly reduced. The vitamin C (Vc) and glutathione (GSH) contents increased significantly by 197.36% and 14.15%, respectively, compared with that of the control. Under chilling stress conditions, 2357 genes from K326 seedlings were differentially expressed significantly compared with that of seedlings grown under the favorable temperature. Of these, 1673 genes were upregulated, and 684 genes downregulated. Gene ontology analysis revealed that these differentially expressed genes were mainly involved in transcription factors, transmembrane transporter proteins, antioxidant systems, signaling pathways, and others. Gene ontology analysis revealed relationships with the photosystem, photosynthetic membrane, chloroplast, plastid, cytoplasm, membrane, ribosomes, and more. In particular, 524 differentially expressed genes were associated with plastid expression. The molecular functions of these differentially expressed genes were mainly associated with oxidoreductase activity and the antioxidant system of the cell membrane of flue-cured seedlings. Differential expression was also observed for genes involved in biological processes such as response to stress, photosynthesis, light reaction of photosynthesis, hormone-mediated signaling pathways, and more. After analysis of metabolism pathways related to differential gene expression, our results showed that genes related to photosynthesis-antenna proteins and photosynthesis were downregulated, while genes involved in oxidant capacity, such as Vc, GSH, and proline metabolism, were upregulated. Leaves of flue-cured tobacco seedlings suffered oxidant damage under chilling stress condition, but the promotion of antioxidant ability by chilling stress had an active protective effect on flue-cured tobacco seedlings. Therefore, flue-cured seedlings had some capacity to adapt to chilling stress by adjustment of gene expression, which altered pathways involved in photosynthetic, oxidant, and antioxidant metabolism to reduce the damage resulting from the stress conditions. Importantly, the results of gene expression analysis and physiological ecological adaptation were consistent. Hence, it is possible to analyze ecological adaptation and differential gene expression of crops under stress, and such studies will facilitate further analysis of gene ontology functions and gene expression relationships.