<p>Glandular trichomes are specialized structures that play a crucial role in plant defense and the production of secondary metabolites. In this study, we investigated the role of <i>NtispH</i>, a key enzyme in the methylerythritol phosphate (MEP) pathway, in regulating glandular trichome density and terpenoid biosynthesis in tobacco (<i>Nicotiana tabacum</i>). Our results showed that over-expression of <i>NtispH</i> led to a significant increase in glandular trichome density, with an average rise of 54.56%. Notably, the density of long-stalked trichomes increased by an impressive average of 131.14%. Additionally, over-expression of <i>NtispH</i> elevated the levels of gibberellin (GA₃). Conversely, RNA interference (RNAi)-mediated silencing of <i>NtispH</i> resulted in reduced trichome density and severe albino phenotypes. Transcriptome analysis revealed that the over-expression of <i>NtispH</i> upregulated a suite of genes involved in chlorophyll biosynthesis, photosynthesis and trichome development, i.e. <i>NtGL2</i>, <i>NtMYB23</i> and <i>NtTTG2</i>, which are homologs of <i>Arabidopsis thaliana</i>. Additionally, the over-expression of <i>NtispH</i> specifically downregulated key genes in the mevalonate (MVA) pathway, namely <i>NtAACT</i>, <i>NtHMGCS</i>, <i>NtHMGCR</i> and <i>NtMVD</i>, while leaving the MEP pathway unaffected. This metabolic shift resulted in significant alterations in the composition of terpenoid volatile organic compounds (VOCs), marked by a significant decrease in the levels of the monoterpene-derived compound trans-β-ionone and a notable increase in the levels of the diterpene neophytadiene. These findings demonstrate that <i>NtispH</i> positively regulates glandular trichome density and may modulate terpenoid biosynthesis by balancing the metabolic flux between the MEP and MVA pathways, providing new insights into the metabolic engineering of tobacco for enhanced stress resistance and economic value.</p>