Abstract: To investigate the role of Abscisic acid (ABA) receptor protein PYL in the promotion of rutin synthesis, a NtPYL6 gene was cloned and identified from Nicotiana tabacum genome, and its role in mediating ABA-induced rutin synthesis was verified by analyzing its sequences and expression patterns. An ntpyl6 mutant was generated and the rutin content in the mutant was determined. The results showed that NtPYL6 was involved in the regulation of ABA-induced rutin synthesis in tobacco. There were two close NtPYL6 genes in the cultivated tobacco genome, named NtPYL6a and NtPYL6b, respectively, which encoded proteins with typical PYR_PYL_RCAR_like domain. The results of gene expression level analysis showed that both NtPYL6a and NtPYL6b exhibited high transcriptional activity in tobacco stalks and leaves, and their expression was significantly induced by ABA, BL, MeJA, and ACC. The results of sequence analysis showed that there were several hormone-related transcriptional regulators in the promoter fragment located at 1 393 bp upstream of the NtPYL6a gene, which could activate the expressions of the above-mentioned genes induced by hormones. The ntpyl6 mutant was generated using the Crispr/cas9 method. Compared with WT plants, the ntpyl6 mutant showed reduced sensitivity to ABA treatment, and ABA-induced rutin synthesis was significantly inhibited in this mutant. Moreover, the ABA-induced expressions of rutin synthesis-related genes were significantly inhibited in the ntpyl6 mutant, and the expression levels of these genes were significantly up-regulated in NtPYL6a overexpression plants. Therefore, NtPYL6 was involved in promoting ABA-induced rutin synthesis and provided a new target gene for tobacco quality improvement breeding.