Abstract: The NtWRKY40 gene encoding a WRKY transcription factor was cloned from tobacco cv. K326 and the gene was subcellularly mapped and overexpressed in tobacco. By comparing the drought resistance of transgenic and control (wild-type) plants, the physiological indexes of drought resistance and the expression of drought stress response genes were determined, and the drought resistance function of the NtWRKY40 gene was analyzed. The results showed that the coding sequence (CDS) of the tobacco NtWRKY40 gene was 777 bp in length, encoding 258 amino acids. Subcellular localization showed that the gene was mainly expressed in the nucleus. Compared with the control plants, the overexpressed transgenic plants of the NtWRKY40 gene exhibited significantly higher drought resistance, the free proline (Pro) content and the superoxide dismutase(SOD)and peroxidase(POD)activities in the transgenic plants were higher, while the malondialdehyde(MDA)content was lower; and the expression levels of NtSOD, NtAPX, NtP5CS and NtNCED1 in transgenic plants were significantly higher under polyethylene glycol (PEG)-simulated drought stress. This study provided preliminary evidence that the NtWRKY40 gene in tobacco could effectively improve drought resistance and provides a research basis for the application of this gene in plant stress resistance genetic engineering.