Abstract: Nicotine synthesis and glandular trichome development are closely related to tobacco quality and resistance, both of which are regulated by jasmonic acid signaling. JAZ1 is a key negative regulator gene in the jasmonic acid signaling pathway. To clarify the effects of NtJAZ1 knockout on nicotine biosynthesis, glandular trichome development and secretion accumulation, the CRISPR/Cas9 technology was used to edit NtJAZ1 in flue-cured tobacco Y2001 and a pure mutant plant HN was obtained. Compared to Y2001, HN tobacco seedlings showed no significant differences in growth and development, with a lower density of glandular trichomes on the leaf surface, but the glandular heads were well developed and fuller than the Y2001, and there were no significant differences in the fractions and contents (mass fraction) of glandular trichome secretions. The nicotine content of tobacco seedlings increased by 19.38% in leaves and 30.85% in roots, and the expression levels of NtMYC2a, NtMYC2b, NtPMT and NtQPT, genes related to nicotine synthesis in roots, increased extremely significantly; aphid resistance of tobacco seedlings was significantly improved. Knockout of the NtJAZ1 gene affected tobacco glandular trichome development, but significantly promoted glandular head development and secretion synthesis, which improved nicotine content and aphid resistance without affecting the leaf surface secretion composition and content. Therefore, the NtJAZ1 gene is an important target for tobacco quality and resistance improvement.