Abstract: In order to clarify the effects of NtJAZ1 gene on nicotine content (mass fraction), growth and development, and quality of tobacco plants, a homozygous mutant NK-9 was obtained by CRISPR/Cas9 technology through the knockout of NtJAZ1 gene in flue-cured tobacco cultivar K326. The sequencing results indicated that the bases of NtJAZ1-1 located between 1 587-1 641 bp and two bases of NtJAZ1-2 located between 1 547-1 548 bp were lost, which generated the premature termination of gene translation. RT-PCR results showed that the expression levels of the key genes, NtMYC2a, NtMYC2b, NtPMT, and NtQPT, in nicotine biosynthesis process increased significantly. The field experiment tests revealed that NK-9 line grew and developed normally, and its plant height, leaf number and leaf width were significantly higher than those of the control (K326), while its leaf length was significantly lower than that of K326. The density of trichome in leaf surface of NK-9 was lower than that of K326 indicated by microexamination. The nicotine content in the middle leaves of NK-9 at flower-bud appearing, topping and mature stages increased by 33.26%, 36.41%, and 39.28% respectively compared with K326. Chemical composition analysis of the cured leaves showed that NK-9 had the balanced chemical compositions and a 44.91% higher content of nicotine than those of K326. The contents of neophytadiene and carotenoid catabolites of neutral aroma components increased significantly, while the contents of browning reaction, phenylalanine catabolites and solanone decreased. All results indicated that NtJAZ1 gene knockout effectively increased the contents of alkaloids in NK-9 and had certain effects on the trichome development and the accumulation of leaf surface chemical components.