Abstract: To investigate the effects of nitrogen application rate on the carbon and nitrogen metabolisms of flue-cured tobacco, field experiments were conducted on cv. Yuyan 10. Three treatments were set as following:low nitrogen (22.5 kg/hm²), moderate nitrogen (26.25 kg/hm²) and high nitrogen (30 kg/hm²). The cell ultrastructure, variations of carbon and nitrogen metabolites and related gene expression patterns of middle leaves of flue-cured tobacco at mature stage were analyzed. The results showed that with the increase of nitrogen application rate, the starch grain volume in cells and the contents of starch and total nitrogen in tobacco leaves increased, while the contents of total sugar and reducing sugar decreased. With the growth of tobacco leaves, AGPase gene (a key gene in carbon metabolism) expression increased, but the increase rate decreased with the increase of nitrogen application rate. NR gene (a key gene in nitrogen metabolism) expression decreased with the increase of nitrogen application rate.