Background and objectives: Phenethyl isothiocyanate (PEITC), a phytochemical from cruciferous vegetables, is known to modulate detoxification enzymes. Fortification of PEITC into a complete nutrition gel, Nutri-PEITC jelly, has been shown to improve its bioavailability. This work aimed to study the effect of Nutri-PEITC jelly on active smokers' detoxification of tobacco-derived carcinogens.Methods: This pre-post trial was conducted on 30 healthy, male, regular smokers. During the pre-intervention period, they smoked regularly for three days. During the postintervention period, they smoked regularly and consumed Nutri-PEITC jelly for three days (40 mg PEITC/day). The total amounts of N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol metabolites in 24-hour urine were measured by liquid chromatography-tandem mass spectrometry after deconjugation with β-glucuronidase and normalized to the urinary creatinine level. To ensure the consistency of smoking and the actual consumption of Nutri-PEITC jelly, the levels of urinary cotinine and PEITC were measured by using an enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry, respectively.Results: After consuming Nutri-PEITC jelly, the level of total urinary NNN metabolites (mainly glucuronide conjugates) was significantly increased by up to 4-fold (p < 0.01). In contrast, the level of total urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol metabolites was not significantly altered (p = 0.325). The urinary cotinine level was similar (p = 0.8832), while the urinary PEITC level was greater than that of the pre-intervention period (p < 0.001). Conclusions:The findings suggest that intake of Nutri-PEITC jelly may increase the detoxification of NNN, a tobacco-specific oral carcinogen, likely by promoting glucuronide conjugation. Future randomized controlled trials are warranted to confirm its poten