Abstract: In order to determine the effect of the mutations in 3-Dehydroquinic acid dehydrase/Shikimic acid dehydrogenase (DHQ/SDH), an important bifunctional enzyme in shikimic acid pathway, on tyrosine synthesis and the release of phenol and other phenolic compounds in cigarette mainstream smoke, fourteen ntdhq/sdh8 mutants were screened and identified by TILLING technology in EMS mutagenesis population of tobacco (Nicotiana tabacum L.). The mutation sites and functional variation of proteins caused by mutation were analyzed, and the mass fraction contents of tyrosine, proline, asparagine and total proteins, as well as the release of phenol, o-dihydroxybenzene, p-dihydroxybenzene, m-dihydroxybenzene, m/p-cresol and o-cresol in cigarette mainstream smoke were determined. The results showed that the tertiary structure of DHQ functional domain in NtDHQ/SDH8 protein was changed due to amino acid variation, which affected the enzymatic activity of the three ntdhq/sdh8 mutants. The tyrosine mass fraction of NtDSM03 and NtDSM12 mutants decreased by 76.78% and 81.71% lower than that of the control, respectively. And phenol release from the cigarette mainstream smoke of the two mutants decreased by 24.09% and 48.99% lower than that of the control, respectively. All of them showed extremely significant difference (P<0.01). Especially, the contents of total proteins, tyrosine, and phenolic compounds e.g. phenol, catechol, m/p-cresol, and o-cresol in cigarette mainstream smoke of NtDSM12 mutant showed extremely significant decrease than wild-type control (P<0.01).