Abstract: The response of stable carbon isotope (δ¹³C) distribution of tobacco cultivars K326, Hongda and KRK26 to climate and environment were studied at rapid growth stage and physiological maturity stage by selecting two ecological tobacco growing areas with nearly four latitude difference, located at Tonghai of Yuxi and Ludian of Zhaotong prefecture in two different climatic zones. The results showed that δ¹³C value of tobacco leaves ranged from -26.56‰ to-24.36‰. The δ¹³C values for all of the tobacco varieties at Tonghai were greater than Ludian, except for KRK26. The δ¹³C value at Ludian was greater than those at Tonghai in maturity stage. In contrast to the rapid growth and maturity stage, the δ¹³C value at maturity stage was greater in all varieties for all locations. With the increasing of maturity of tobacco leaves, water use efficiency of the tobacco gradually increased in two climatic zones. Comparison between varieties, for both growth stages, the δ¹³C value of KRK26 was the largest in Tonghai, the δ¹³C value of Hongda was the largest in Ludian. The average value of δ¹³C in tobacco of the lower leaves and cutters in Tonghai was greater than Ludian, which support the conclusion that the higher temperature, the longer sunshine hours, the less precipitation, the value of δ¹³C was larger. The results indicate that temperature, sunlight, precipitation, and variety have significant effect on tobacco δ¹³C value.