Abstract: In order to investigate the effects of exogenous 2, 4-epibrassinolide (EBR) on nitrogen metabolism and nicotine metabolism in flue-cured tobacco at mature stage, the differences of photosynthetic characteristics, dry matter accumulation, activities of nitrogen metabolism related enzymes, relative expression levels of nicotine synthesis related genes at mature stage, and chemical components of flue-cured tobacco were compared between the treatments of applying exogenous EBR by foliar spraying(T1)and root irrigation(T2), with clear water as the control(CK)after topping. The results showed that compared with CK, applying exogenous EBR by foliar spraying and root irrigation could improve the photosynthetic capacity of mature tobacco plants, increase the dry matter accumulation in various parts of tobacco plants, and significantly increase the contents of total nitrogen and nicotine in cured middle and upper tobacco leaves. The effects of foliar spraying were better than that of the root irrigation applying treatment. The activities of the key enzymes of nitrogen absorption and assimilation, NR and GS, in the upper leaves of the two treatments, were significantly higher than those of CK. The relative expression levels of nitrogen assimilation related genes NtGS2, NtGDH1 and NtNR in the leaves of T1 were significantly higher than those of CK and T2. The relative expression levels of nicotine synthesis genes NtQPT and NtPMT, and transport genes NtJAT1, NtJAT2 and NtNUP1 in different parts of tobacco plants under the two EBR application treatments were significantly higher than those of CK, and the expression levels of nicotine synthesis and transport genes in leaves of T1 were significantly higher than those of CK and T2. Therefore, exogenous EBR could promote the growth and development of tobacco plants, improve the levels of nitrogen metabolism and nicotine metabolism and increase the nicotine content in cured middle and upper tobacco leaves, and the effects of foliar spraying treatment was better than those of the root irrigation treatment.