Abstract: To investigate the biological function of hydroperoxide lyase (HPL) in tobacco resistance to adversity stress, a hydroperoxide lyase gene was cloned from Nicotiana tabacum K326 and designated NtHPL1. Bioinformatic analysis was conducted on the NtHPL1 protein, and the relative expression level of the NtHPL1 gene was analyzed by qPCR method. The function of NtHPL1 gene in stress resistance was predicted by creating transgenic materials with NtHPL1 overexpression. The results showed that: 1) The total length of the coding region of NtHPL1 gene encoding 454 amino acids was 1 365 bp. The results of protein structure and phylogenetic analysis showed that NtHPL1 belongs to 13-HPL subfamily. 2) The NtHPL1 gene was expressed in tobacco roots, stalks, leaves, flowers and axillary buds, with the relative expression level in flowers being the highest. The relative expression level of NtHPL1 gene in tobacco lcaves varied at different developmental stages, with the lowest expression level at full bloom stage and the highest expression level at maturity stage. Both salt and drought stress could induce the expression of NtHPL1 gene. 3) After salt and drought stress treatments, the leaves of wild-type tobacco turned yellow, while the NtHPL1-overexpressed tobacco grew normally. The chlorophyll and proline contents (mass fractions) in leaves of NtHPL1-overexpressed tobacco were higher than those of wild-type tobacco, and the malondialdehyde content was lower than that of wild-type tobacco, indicating that overexpression of the NtHPL1 gene could improve the resistance of tobacco to salt and drought stress.