Burley tobacco (Nicotiana Tabacum) is a chlorophyll-deficiency mutant. Nitrate is one precursor of tobacco-specific nitrosamines (TSNAs) and is largely accumulated in burley tobacco. To decrease nitrate accumulation in burley tobacco, glycerol, a polyhydric alcohol compound and physiological regulating material, was sprayed and its effects were investigated based on metabolomic technology and molecular biology. The results showed that glucose, glutamine and glutamic acid increased by 2.6, 5.1 and 196, folds, respectively, in tobacco leaves after glycerol application. Nitrate content was significantly decreased by 12-16% and expression of eight genes responsible for carbon and nitrogen metabolism were up-regulated with glycerol applications under both normal and 20% reduced nitrogen levels (P < 0.01). Leaf biomass of plants sprayed with glycerol and 20% nitrogen reduction was equivalent to that of no glycerol control with normal nitrogen application. Carbohydrates biosynthesis, nitrate transport and nitrate assimilation were enhanced in glycerol sprayed burley tobacco seedlings which might contribute to reduced nitrate and increased carbohydrates contents. In conclusion, glyerol spray coupled with 20% nitrogen reduction would be an effective method to reduce nitrate accumulation in burley tobacco.