Abstract Nicotine concentration is a key index and directly affects the industrial quality and availability of flue-cured tobacco (FCT). Seedlings transplanted at different times were subjected to different climatic conditions, which were closely correlated with the growth, development, and nicotine synthesis of FCT. An appropriate transplanting time is imperative for ensuring high-quality tobaccos. Hence, a 15N tracing experiment in the field (which included three treatments: FCT seedlings transplanted on 5th, 15th, and 25th May) was carried out to evaluate the influence of different transplanting dates on 15N utilization and nicotine concentration in FCT leaves. Results showed that compared to the tobacco seedlings transplanted on 5th May, the seedlings transplanted on 15th and 25th May increased the dry matter weight by 9.86 - 87.5 % and N uptake by 24.4 - 36.9 % in shoots during the field growing periods of FCT plants. However, it was notable that postponing the transplanting time of FCT seedlings decreased the proportion of nicotine-N to total N (26.9 - 50.6 %), 15N abundance in total N (7.27 - 40.7 %), and nicotine-15N abundance in total nicotine-N (7.30 - 35.7 %), thus inducing a noticeable reduction of 24.3 - 35.8 % in the nicotine concentration in FCT leaves. Hence, delaying transplanting time promoted dry matter and N accumulation, while it significantly decreased the nicotine concentration in FCT leaves, which is of great importance in improving the industrial quality and availability of tobaccos.