Abstract: To verify the function of NtHDZIV8 in the development of glandular trichomes, the coding sequence (CDS) of NtHDZIV8 was cloned from Honghuadajinyuan and further used in bioinformatics analysis. The expression pattern of NtHDZIV8 was analyzed by qRT-PCR, and the role of NtHDZIV8 in development of glandular trichomes was verified using subcellular localization and virus-induced gene silencing (VIGS). The results showed that NtHDZIV8 was highly conserved and contained typical HD-LZ, START and SAD domains, belonging to the HD-ZIP IV family. The expression pattern analysis showed that NtHDZIV8 was highly expressed in glandular trichomes, and was induced by plant hormones GA3, NAA and MeJA. Subcellular localization results showed that NtHDZIV8 was located in the nucleus. The results of VIGS analysis showed that NbHDZIV8 gene regulated basal expansion of stalk cells in long glandular trichomes, but did not affect the morphology and density of short glandular trichomes in N.benthamiana. The results in this study demonstrate that NtHDZIV8 regulates the development of long glandular trichomes in tobacco, which provides a theoretical basis for the analysis of the regulatory mechanism of tobacco trichome development.