Abstract: To determine the effects of mutations in 3-dehydroquinic acid dehydrase/shikimate dehydrogenase (DHQ/SDH), an important bifunctional enzyme in the shikimate pathway, on tyrosine synthesis and the release of phenol and other phenolic compounds in mainstream cigarette smoke, fourteen ntdhq/sdh8 mutants were screened and identified by TILLING technology in the EMS mutagenesis population of tobacco (Nicotiana tabacum L.). The mutation sites and the functional variation of the proteins caused by the mutation were analyzed, and the mass fractions of tyrosine, proline, asparagine, and total protein, as well as the release of phenol, o-dihydroxybenzene, p-dihydroxybenzene, m-dihydroxybenzene, m/p-cresol, and o-cresol in mainstream cigarette smoke were determined. The results showed that the tertiary structure of the DHQ functional domain in NtDHQ/SDH8 protein was altered by amino acid variation, which affected the enzymatic activity of the three ntdhq/sdh8 mutants. Compared with the wild-type control, the tyrosine mass fraction in NtDSM03 and NtDSM12 mutants decreased by 76.78% and 81.71%, respectively; and the phenol release from mainstream cigarette smoke of the two mutants decreased by 24.09% and 48.99%, respectively; all of which showed extremely significant differences (P < 0.01). In particular, the mass fractions of total protein, tyrosine, and phenolic compounds such as phenol, catechol, m/p-cresol, and o-cresol in the mainstream cigarette smoke of the NtDSM12 mutant decreased extremely significantly compared to the wild-type control (P < 0.01).