Abstract: In order to investigate the effects of transplanting date (5th May, 15th May, 25th May) of flue-cured tobacco (FCT) seedlings and nitrogen fertilization on nicotine synthesis and sources of nicotine N of FCT, a field experiment with 15N isotope tracing micro-plots was carried out in Laowan (N 3127, E 11114, 1130 m above sea level), a main tobacco production area of Xiangfan city, Hubei province. The results showed that, compared with the transplanting date of 5th May, postponing the transplanting date to 15th-25th May increased the nicotine concentrations, the proportion of nicotine N to total N, and the proportion of nicotine fertilizer N in organs of FCT plants by 10%-85%, 5%-110%, and 21%-56%, respectively. Applying N fertilizer significantly increased the nicotine concentration and the proportion of nicotine N to total N by 12%-59% and 5%-127%, respectively. The proportion of nicotine N to total N in different parts of FCT plants increased with growing of the plants, and the proportion of nicotine fertilizer N decreased with the growing of FCT plants and the rising of leaf position. Soil N was the main source of nicotine N. These results suggested that decreasing of N supplication and early transplanting make for reducing of nicotine concentrations in FCT leaves, especially in the middle and upper leaves.