Abstract: To explore the regulatory network of NtHD9 involved in the formation of long glandular trichomes (LGTs), analyses were conducted on NtHD9-SM, NtHD9-DM, NtHD9-OE plants. The expression levels of genes involved in jasmonic acid (JA) synthesis, metabolism and signal transduction were examined in wild-type (WT) and NtHD9 transgenic plants. Results revealed that both the size of glandular heads in LGTs and JA content followed the same order across the four lines: NtHD9-OE>WT>NtHD9-SM>NtHD9-DM. With increasing concentrations of methyl jasmonate (MeJA), the densities and sizes of LGT glandular heads in NtHD9-SM and NtHD9-DM plants increased, with head morphology normalizing at 200 μmol/L MeJA. RT-qPCR analysis revealed that NtHD9 expression was positively correlated with JA synthesis-related gene (LOX1, AOS and AOC2), JA metabolic gene (JAR1), JA signal transduction gene CYP94C1, several NtJAZ family members (NtJAZ3, NtJAZ5 and NtJAZ7), and NtHD-ZIP Ⅳ family members (NtHD3, NtHD7, NtHD9, NtHD11 and NtHD12). These genes exhibited decreased expression in NtHD9-SM and NtHD9-DM plants, while increased expression in NtHD9-OE plants. Conversely, three JA signaling-related genes (WRKY62, WRKY68 and NtJAZ12) showed the opposite trend, showing increased expression in NtHD9-SM and NtHD9-DM plants, and decreased expression in NtHD9-OE plants. These results indicatethat NtHD9 positively regulates the formation of glandular head in LGTs through JA signaling pathway in tobacco.