Abstract: NAC family is one of the largest transcription factor families in plants, and plays a significant regulatory role in plant development, signal transduction and abiotic stress response. Cloning and validation of NAC related genes in tobacco can provide a theoretical basis for tobacco breeding for stress tolerance. In this study, NtNAC055 was cloned from root cDNA of cv. K326 by RT-PCR. In addition, bioinformatics analysis, expression pattern analysis, subcellular localization and transactivation activity analysis of NtNAC055 were performed. The results showed that the full-length of NtNAC055 was 1032 bp, and the NtNAC055 protein was 343 aa that contained a typical NAC domain. Phylogenetic analysis indicated that NtNAC055 had high homology with ANAC055 from Arabidopsis. Subcellular localization and transactivation analyses showed that NtNAC055 was localized in the nucleus and had transactivation activities. The expressions of NtNAC055 were analyzed by qRT-PCR in different tissues of tobacco, and in seedlings under different abiotic stresses. The results showed that NtNAC055 was expressed in tobacco roots, stems, leaves, flowers and axillary buds, especially in roots. The expression levels of NtNAC055 were induced under drought and heat stresses, which indicated that NtNAC055 might be involved in tobacco abiotic stress responses.