At the present study, the tobacco variety Yunyan 87 was used as the experimental material, and different growth temperatures were simulated in artificial climate chambers to resemble the natural climates of three areas (Jiangchuan, Zunyi, and Xiangxian), to investigate the effects of different growth temperature on pyridine alkaloids contents (nicotine, nornicotine, anatabine, anabasine) and nicotine metabolism in tobacco, at three different growth stages as follows: the transplanting to resettling stage, resettling to squaring stage, and maturity stage. The results indicated during all three selected stages, the biosynthesis and accumulation of nicotine in roots were stimulated by a lower growth temperature. During the transplanting to resettling stage, the highest level of nicotine content under a lower temperature reached to 1.54-fold of that at the initial level (0 d), accompanied by a much lower nicotine content in leaves. When grown to thesettling to squaring stage and maturity stage, a higher temperature was helpful to the accumulation of nicotine in leaves, which was approx. 1.80-4.38 fold higher than the initial level. In addition, the profiles of anatabine, anabasine and nornicotine contents quite resembled those of nicotine. In general, at different growth stages, the biosynthesis and distribution of nicotine were deferentially regulated by growth temperature, while the temperature at mature stage performed the greatest effect on nicotine content. Thus Xiangxian, which had higher growth temperature than the other two regions, was more suitable for nicotine accumulation in tobacco leaves. These results revealed the mechanism underlying the effects of temperature on regulation of alkaloid contents and nicotine metabolism in tobacco, and to some extent, explained why tobacco leaves from these three regions exhibit great differences in flavor style based on fluctuations of nicotine content.