Abstract: To elucidate the biological function of NtTRY in regulating glandular trichome development in tobacco, the NtTRY gene was cloned from Nicotiana tabacum Honghua Dajinyuan. Comprehensive bioinformatic analyses were performed to characterize its conserved domains and gene structure. Its regulatory function in glandular trichome development was investigated through subcellular localization, quantitative real-time PCR (qRT-PCR), virus induced gene silence (VIGS) and overexpression assays. The results indicated that the full-length coding sequence (CDS) of NtTRY is 267 bp, encoding a protein of 88 amino acids. of the encoded protein contains a typical R3-MYB domain and is localized in the nucleus. The qRT-PCR analysis revealed that NtTRY is highly expressed in roots, while its expression is relative lower in young leaves and glandular trichomes. In NbTRY VIGS plants, itsexpression was significantly downregulated, accompanied by increases in glandular trichome density of 26.98% and 35.65%, respectively.In contrast, T1 transgenic plants overexpressing NbTRY exhibited a significant reduction in glandular trichome density, with an average decrease of 29.67%. Notably, the OE-2 line exhibited the most pronounced reduction(31.33%). In conclusion, these findings support that NtTRY negatively regulates glandular trichomes density in tobacco. This research provides a theoretical foundation for the genetic regulation of glandular trichomes development and identifies a candidate gene for improving tobacco quality and stress resistance through molecular breeding.