Abstract: To elucidate the biological function of NtNAC029 gene in the regulation of leaf senescence and flavonoid biosynthesis in tobacco, a NAC transcription factor gene (NtNAC029) was cloned from cultivated tobacco K326 based on information from the homologous gene in Arabidopsis thaliana. The coding region and the encoded amino acids of this gene showed conserved sequence structures and protein domains of the NAC family. The expression of the NtNAC029 gene showed significant tissue and stage specificities. Its expression level in leaves gradually increased with leaf maturity and senescence, and reached its maximum value at the late stage of leaf senescence. However, its expression level in capsules increased sharply at the early stage of development and then gradually decreased, indicating that this gene may be involved in regulating important physiological processes such as tobacco leaf senescence and fruit development. The expression of NtNAC029 gene was more sensitive to light, and the gene expression level was significantly decreased under light culture conditions, while darkness treatment significantly upregulated the gene expression level. In addition, salt, abscisic acid (ABA) and methyl jasmonic acid (MeJA) could significantly induce the expression of NtNAC029 gene, indicating that NtNAC029 may play an important role in the response to multiple environmental factors in the regulation of tobacco leaf senescence.