Aimed to explore the ecological reasons for the difference in nicotine content of flue-cured tobacco planted in different regions of Hunan Province, field experiments were conducted in Sangzhi, Liuyang, and Yongzhou counties, the three typical tobacco regions of Hunan Province, taking tobacco variety K326 as the test object. Simultaneously, pot experiments with local soils and guest soils were carried out. The nicotine content of mid position tobacco leaves was analyzed at harvest time. Field experiments showed that the average nicotine content of tobacco leaves differed significantly among test sites, with that in Sangzhi being the highest, followed by Liuyang, and Yongzhou. Pot experiments showed that climate had significant effects on the average nicotine content of tobacco leaves, while soil and its interaction with climate had less effects. The contribution rate of climate, soil, and their interaction on the variance of the average nicotine content was 60.0%, 12.8% and 27.2%, respectively. The main sub-ecological factors closely related to the average nicotine content of flue-cured tobacco planted in different regions of Hunan Province were in turn the cloud cover at maturing stage, the relative humidity, sunshine hours, diurnal temperature variance, and rainfall at root-extending stage, and the average air temperature at vigorous growth stage. Generally, climate was the main ecological factor that led to the nicotine content difference of flue-cured tobacco planted in different regions of Hunan Province.