Abstract: CRISPR/Cas9 is an important technology, which has been widely used in targeted gene editing and creation of molecular breeding materials. In order to generate tobacco materials with low nicotine levels for molecular breeding, the flue-cured tobacco variety K326 was used as materials in this study, and genome editing technology was carried out to edit 5 genes (PMT1, QPT1, A622, NtNUP1 and JAT1) controlling nicotine synthesis and transport in tobacco. Finally the nicotine contents of T2 plants with homozygous genotypes were determined. The results showed that a total of 100 T0 transformed plants with targeted gene knockout successfully were obtained. The mutation rate was 29.9% and the main mutation type was single base insertion or deletion. The QPT1 and JAT1 gene sequences of T1 plants (knockout successfully) with homozygous genotypes were analyzed. There were four homozygous mutations that inserted a single T, C and A base, and one homozygous deletion mutation of 44 nucleotides, leading to the amino acid chains greatly shortened by frameshift mutation. Moreover, the tobacco nicotine contents in the upper leaves of the T2 mutants, which were from selfed T1 mutants, were significantly lower than that in wild type plants. Therefore, the results indicated that CRISPR/Cas9 editing technology could effectively mutate genes controlling nicotine synthesis and transport in tobacco, which provided theoretical and technological support for molecular breeding for low-nicotine tobacco.