Abstract: This study used the mutant and its wild type as materials, and the physiological, biochemical, and metabolic differences of the mutant were analysed by measuring agronomic traits, glandular hair density, Cembranoid diterpenes and its metabolic precursor content, and the expression level of key genes involved in Cembranoid diterpenes metabolism. Combined with gene expression differences, the physiological and molecular mechanisms of the mutation were preliminarily analyzed. The results showed that the content of Cembratrien-ol and Cembratrien-diol in the mutant was 2.63 times and 4.03 times higher than that of the wild type, respectively, both significantly higher than that of the wild type. The density of secretory glandular hairs were 757.38 strips/cm~2, very significantly higher than that of wild type. The metabolic precursors of Cembranoid diterpenes in the mutant were 849.83 ng/g and 298.29 ng/g, respectively, with no significant difference from the wild type, The expression levels of the mutant Cembratrien-ol synthase gene NtCYC-1 and Cembratrien-diol synthase gene NtCYP71D16 were 17.77 and 20.89 times higher than those of the wild type, respectively, both significantly higher than those of the wild type. Study showed that mutant was an excellent material for breeding high-quality, insect-resistant flue-cured tobacco varieties. The up-regulated expression of the NtCYC-1 and NtCYP71D16 genes in the mutant improved the synthesis of Cembratrien-ol and Cembratrien-diol. At the same time, the higher density of secretory glandular hairs in the mutant provided more sites for the synthesis of Cembranoid diterpenes, resulting in a significant increase in the content of Cembranoid diterpenes.