Abstract: To elucidate the biological function of NtTRY in regulating glandular trichome development in tobacco, the NtTRY gene was cloned from Nicotiana tabacum Honghuadajinyuan. 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 silencing (VIGS) and overexpression assays. The results indicated that the full-length coding sequence (CDS) of NtTRY was 267 bp, encoding a protein of 88 amino acids that contains a typical R3-MYB domain and was localized in the nucleus. The qRT-PCR analysis revealed that NtTRY was highly expressed in roots, while its expression was relatively lower in young leaves and glandular trichomes. In NbTRY VIGS plants, the target gene expression 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 trichome density in tobacco. This research provides a theoretical foundation for the genetic regulation of glandular trichome development and identifies a candidate gene for improving tobacco quality and stress resistance through molecular breeding.