Abstract: To explore the driving effects of climate factors on nicotine accumulation in flue-cured tobacco and its future trends, this study, based on 5,321 tobacco leaf samples and corresponding meteorological data from three typical tobacco-growing regions in China (Southwest, Huanghuai, and Northeast) from 2010 to 2024, comprehensively utilized variance partitioning, correlation analysis, and ARIMA time series models. It systematically dissects the spatiotemporal evolution characteristics of nicotine content, climate-driven effects, and cultivar ecological adaptability. The results indicated that over the past decade, nicotine content across all stalk positions in the Southwest region exhibited an overall increasing trend, with upper and middle leaves increasing by 20.3% and 16.7%, respectively. Conversely, the Huanghuai and Northeast regions displayed an overall decreasing trend. The climate-driven effects demonstrated high stalk position specificity; nicotine in the upper leaves was primarily regulated by sunshine duration (with a contribution rate of 42.5%), whereas nicotine in the middle and lower leaves was predominantly driven by temperature and precipitation. Temporally, sunshine duration in the Southwest region decreased significantly at a rate of 2.1 hours per year (p < 0.05), while precipitation in the Huanghuai and Northeast regions increased annually by 21 mm and 17 mm, respectively. Predictions based on the ARIMA model indicate that, driven by climatic fluctuations over the next five years, nicotine content in the Southwest tobacco region is projected to reach 2.9%–3.1% for upper leaves and 2.1%–2.2% for middle leaves. In contrast, nicotine levels in middle leaves from the Huang-Huai region are expected to remain steady at 2.1%, while upper leaves in the Northeast region will likely fluctuate within the range of 1.8%–2.1%. The response of nicotine to climatic factors exhibited significant genotype specificity. Cultivar Yunyan 87 was highly sensitive to sunshine and precipitation, whereas cultivars such as Yunyan 99, Zhongyan 100, Yunyan 97, and Longjiang 911 were insensitive to climate fluctuations, demonstrating superior climate resilience. This study elucidates the climate-driven pathways of nicotine formation in typical tobacco-growing regions, providing a crucial scientific basis for for the early diagnosis of tobacco leaf raw materials, proactive industrial responses, and the rational spatial layout of elite germplasm under the context of climate change.