Abstract: A new flue-cured tobacco line(coded Hc400 was developed by silencing the NtDHS gene in cv. K326)tends to exhibit mottled yellowing at the seedling stage. To address this issue and elucidate the effects of light quality on the growth of this flue-cured tobacco seedlings, K326 and Hc400 were selected as experimental subjects. Four combinations of light quality, high red light(red light ∶ blue light = 3 ∶ 1), balanced light(red light ∶ blue light = 1 ∶ 1), high blue light(red light ∶ blue light = 1 ∶ 3)with white light as a control (illumination light), were established to investigate the impact of different light quality combinations on the growth and physiological characteristics of the flue-cured tobacco. The results showed that under the high red light treatment, the activities of phosphoribulokinase (PRK), ATP synthase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribulose-1, 5-bisphosphate carboxylase/oxygenase(Rubisco)in Hc400 leaves increased by 4.20%, 41.13%, 25.20% and 56.98%, respectively, compared to the control. The transpiration rate and net photosynthetic rate increased by 13.48% and 58.82% respectively and the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids increased by 54.07%, 63.64%, 56.84% and 59.09% respectively. In addition, the chloroplasts were more densely arranged and had a fuller and rounder shape. Under the high red light treatment, the chloroplasts of cv. K326 showed increased volume and were more densely arranged compared to those under the white light control, resulting in significantly improved agronomic traits and enhanced photosynthetic rates. These results suggest that high red light irradiation can be used in production to reduce mottling and yellowing in seedlings of the new line Hc400, while maintaining the desirable traits of the conventional cv. K326.