Abstract: As the front line of biological and abiotic defense, and the site of secretion and synthesis of secondary metabolites, regulation of tobacco glandular hair is of great importance for tobacco stress resistance and quality breeding. To explore the function of MYB transcript factors in tobacco glandular trichomes development, one MYB transcription factor NtMYB17 gene were cloned from common tobacco in this study. The analysis involved examining the conserved domain, assessing tissue-specific gene expression via qRT-PCR, and conducting a subcellular localization to further scrutinize the expression pattern of the gene. Results showed that NtMYB17 had a total length of 939 bp, encoding 312 amino acids, a typical MYB domain and was localized in the nucleus at the subcellular level. qRT-PCR revealed that the expression of NtMYB17 in glandular trichomes was eight times higher than that in trichome-free leaves. The expression level was higher in flower buds and young leaves, and it was lowest in roots. Following the overexpression of NtMYB17 in tobacco, an increase was observed in the number of branch-type glandular trichomes. This study indicated that NtMYB17 played a regulatory role in the morphology of tobacco glandular trichomes.