Abstract: 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme in the synthesis of terpenoids. To reveal the potential function of DXS2 in tobacco isoprenoid biosynthesis, NtDXS2 was cloned from Nicotiana tabacum by Rapid Amplification of cDNA Ends (RACE) technology. The sequence characteristics and expression patterns of NtDXS2 in different tissues and salicylic acid treated leaves were analyzed. The results showed that the full-length cDNA of NtDXS2 was 2 407 bp, which encoded 713 amino acids. Amino acids sequence of NtDXS2 matched that of tomato DXS2 and NtDXS1 with the identity of 91.1% and 69.2%, respectively. The prediction results of subcellular localization and protein domain indicated that NtDXS2 might be located in chloroplast and contained the conserved domains of DXS. Phylogenetic analysis suggested that NtDXS2 belonged to DXS2 subfamily and had closest genetic relationship with tomato DXS2. qPCR revealed that the expression levels of NtDXS2 were higher in leaves, lower in roots and stamens, and hardly detected in pistils and sepals. The expression level of NtDXS2 increased under salicylic acid treatment. Therefore, NtDXS2 might participate in the terpenoid synthesis in glandular trichomes of tobacco leaves and the reaction against disease of tobacco.