Nitrogen stress is the main abiotic stress factor affecting the carbon and nitrogen balance of flue-cured tobacco. Tandem mass tag (TMT) proteomics was used to compare the differently expressed proteins (DEPs) of flue-cured tobacco under high- (HN) and low- (LN) nitrogen stress, and Gene Ontology (GO) function annotation, Kyoto Encyclopedia for Genes and Genomes (KEGG) analysis, and protein-protein interaction (PPI) analysis were conducted. The results showed that the difference between LN and HN stress was mainly reflected in the formation pathways of carbon and nitrogen metabolism.