Background: Nitrosition of tobacco pyridine alkaloids by nitrate-derived NOx is the origin of tobacco-specifc nitrosamines (TSNAs) formed in tobacco, which are among the most notable toxicants present in tobacco products and smoke. Burley tobacco is particularly susceptible to TSNA formation because the cultivars exhibit a chloroplast-deficient and nitrogen-use-defciency phenotype which results in high accumulation of nitrate. Strategies to lower nitrate levels in tobacco could produce a corresponding decrease in TSNAs accumulation in leaves. Previous studies have showed that grafting with high nitrogen use efficiency rootstock was able to improve the nitrogen utilization of flue-cured tobacco. In this study, a reciprocativel grafting experiment has been conducted with two varieties of different tobacco types (burley tobacco ‘Eyan No.1’ and flue-cured tobacco ‘K326’) to investigate whether replacing burley tobacco root with flue-cured tobacco by grafting can enhance pigment biosynthesis and photosynthesis parameters and reduce nitrate content of burley tobacco leaves, and to explore the corresponding mechanism. Results: The results showed that compared with the self-rooted burley tobacco seedlings, grafting significantly increased the pigment content, net photosynthesis, biomass, total soluble sugar, reducing sugar, nitrate reductase activity, glutamine synthetase activities, NH4-N and soluble protein content of the leaves of Eyan No.1 while decreased the nitrate content and NO3-N/TN. Interestingly, transcription level analysis suggested that replacing burley tobacco root with flue-cured tobacco by grafting was able to up-regulate the genes involved in starch and sucrose metabolism, porphyrin and chlorophyll metabolism, carbon fixation in photosynthetic organism metabolism, carotenoid biosynthesis and nitrogen metabolism of burley tobacco leaves. In addition, the PPI network revealed gene_68511, gene_35043 and gene_77508 had higher degrees via replacing burley tobacco root with flue-cured tobacco, which might be hub proteins to reduce nitrate accumulation. Conclusions: In conclusion, grafting with high nitrogen use efficiency rootstock provided an exceptionally promising means of nitrate reduction in burley tobacco leaf , which is a principle precursor of TSNAs, due to the improvement of photosynthesis and nitrogen metabolism in the scion.