Water is a major ecological factor for the growth, physiological metabolism and quality formation of tobacco. Drought causes not only the decrease of tobacco yield and quality, but also the lowering of nitrogen utilization rate, leading to nitrate accumulation and increased levels of tobacco specific nitrosamine in cured and stored leaves. Salicylic acid, a phenolic compound and signal substance, is involved in regulating many plant physiological processes. The flue-cured tobacco variety K326 and Burley tobacco variety TN90 were used in the pot experiments to investigate the physiological responses of tobacco to salicylic acid under PEG drought stress and natural drought stress conditions. The pigment contents, photosynthetic characteristics, key nitrogen metabolism enzyme activities, antioxidant enzyme activities, nitrate contents etc., were measured after 0.3 mmol·L^-1 SA treatment. Transcriptome sequencing and GO/KEGG analysis were also performed. The results showed that under drought stress conditions, the photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) decreased significantly, while the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and the contents of malondialdehyde (MDA), proline and nitrate were all increased. Compared with PEG drought stress (D), SA spray significantly improved all the physiological parameters, with the Pn, Gs and Tr increasing by 45.74 %, 26.82 %, 52.22 %, and the activities of SOD, POD, CAT, protein and proline content increasing by 44.27 %, 50.18 %, 25 %, 34.69 % and 24.88 %, respectively; and nitrate content was reduced significantly. Compared with natural drought stress, SA treated leaves had higher Pn, Gs, Tr and Ci of 23.41 %, 46.88 %, 50.93 % and 52.27 %, respectively; POD activity and proline contents also increased, while MDA content decreased; nitrate reductase (NR) and glutamine synthetase (GS) activities increased by 47.08 % and 23.63 %, and nitrate content decreased by 12.98 % over the drought stress control. GO and KEGG analysis showed that SA treatment was able to up-regulate the genes involved in photosynthesis (RCA1, PSBO, PSBR, PSAO), photosynthesis-antenna proteins (lhcA-P4, LHC4.2, CAB50), carbon metabolism (FBP, FBA1, RBCS), porphyrin and chlorophyll metabolism (POR1, hemC, HEMA1, CHLM) and starch and sucrose metabolism (SS1, bglX, AGPS1). The conclusion is that SA application would effectively improve the photosynthesis of tobacco under drought conditions, thus increase nitrogen use efficiency and reduce the accumulation of nitrate which is the precursor of TSNA.