Abstract: Field survey and laboratory analysis were conducted to determine the distribution and relationship of soil nitrogen and flue-cured tobacco nicotine content in representative locations in Xuan’en and Xianfeng of Enshi tobacco cultivation areas. The results show a suitable total nitrogen content and a markedly higher available soil nitrogen content in Enshi tobacco cultivation areas. Based on soil nitrogen content, 129 soil samples are divided into three groups of high, medium and low nitrogen content via cluster analysis. Samples with low nitrogen content account for 60.47% of the 129 soil samples. Flue-cured tobacco nicotine content ranges from 8.18 g·kg^-1 to 42.25 g·kg^-1, with a mean and variation coefficient of 22.80 g·kg^-1 and 36.42% respectively. Samples with nicotine content in the range of 15 g·kg-1 to 35 g·kg^-1 account for 66.94% of the total. Significant differences exist in nicotine content of flue-cured tobacco from different cultivars, gradings and altitudes. Flue-cured tobacco nicotine content occurs in the following trend of “K326” > “Yunyan85” > “Yunyan87” for cultivars; B2F > C3F > X2F for grades; and low altitude > medium altitude > high altitude for altitudes. A regression relationship exists between flue-cured tobacco nicotine and soil nitrogen content. However, F-test shows that the relationship is insignificant at 5% confidence level.