Abstract: To investigate the responses of tobacco roots to nitrate signals, taking flue-cured tobacco variety K326 as experimental material, seedling culture and nitrogen stress experiments were conducted in a greenhouse with controlled light, temperature and humidty (14 h light, 10 h dark, temperature 28℃, relative humidity 65%-75%) and transcriptome sequencing technique was used to study the differentially expressed genes (DEGs) in tobacco roots at high (NO₃^- 5.0 mmol/L) and low (NO₃^- 0.1 mmol/L) levels of nitrate nitrogen applying after 0, 6, 12 and 24 h. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that the expressions of some DEGs in sub-cluster increased first and then decreased at high and low levels of nitrate nitrogen applying after 6 h. These genes were mapped in phenylpropanoid biosynthesis and the metabolic pathways of alanine, aspartic acid and glutamic acid. The results indicated that the metabolism pathways of amino acids and carbon/nitrogen were important to respond nitrogen signal. However, some genes were involved in the biosynthesis of tropone, piperidine and pyridine alkaloid. The metabolism pathways of pyrimidine, purine, fructose, mannose, starch and sucrose down-regulated at the low level of nitrate nitrogen while they up-regulated at the high level of nitrate nitrogen, which suggested that the alkaloid synthesis and energy metabolism in flue-cured tobacco roots were inhibited at the low level of nitrate nitrogen and were promoted at the high level of nitrate nitrogen.