Nitrogen is one of the important nutrients for plants. Nitrate is also one of the important signal molecules for plant growth. In order to elucidate how the tobacco root responds to outside nitrate-nitrogen signals, a transcriptome sequencing technique was used to study the profiles of gene expression in tobacco roots at 0 h, 6 h, 12 h, and 24 h after exposure to high and low nitrate treatments. KEGG pathway analysis indicated that the gene expression profiles in tobacco roots 6 h after being exposed to high and low nitrogen nutrients were pivotal and included many common as well as different expressed genes. Some genes in the roots were simultaneously up-regulated after the 6 h treatment at high and low levels of nitrogen nutrition and then decreased in both groups. These genes were mainly involved in phenylpropanoid biosynthesis, alanine, aspartate and glutamate metabolism pathways. Some important genes such as glutamate dehydrogenase, glutamic acid decarboxylase and asparagine synthetase which were involved in the carbon and nitrogen metabolism were up-regulated. Results also indicated that amino acids and carbon/nitrogen metabolism pathways were vital metabolic pathways that responded to outside nitrogen nutrition and may be the common signal pathways in root physiological activities no matter what the nitrate level. However, some genes involved in tropane, piperidine and pyridine alkaloid biosynthesis, pyrimidine metabolism, purine metabolism, fructose and mannose metabolism, and starch and sucrose metabolism pathways were down-regulated at low-nitrogen levels and were up-regulated when nitrogen levels were high. Results suggested that low levels of nitrate nitrogen could lower the alkaloid synthesis and energy metabolism in the root of flue-cured tobacco while high levels of nitrate nitrogen nutrition could promote these metabolic pathways.