2-Phenethyl isothiocyanate (PEITC) is a natural product found as a conjugate in cruciferous vegetables. It has been reported to have preventative properties against lung cancer and to inhibit metabolic activation of tobacco carcinogens. In this study, we evaluated the ability of PEITC to influence the metabolism of the human carcinogen 1,3-butadiene in current smokers in a phase II clinical trial with a crossover design. Urinary mercapturic acids of 1,3-butadiene were quantified at baseline and during PEITC treatment. Seventy-nine smokers were randomly assigned to one of two arms: PEITC followed by placebo or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. Oral ingestion of PEITC increased urinary levels of BD-mercapturic acids (MHBMA and DHBMA) by 11.1% and 3.7%, respectively, but these increases were not statistically significant (P ¼ 0.17 and 0.64, respectively). A much stronger effect was observed among subjects with the null genotype of both GSTM1 and GSTT1: in these individuals, PEITC increased urinary levels of MHBMA by 58.7% (P ¼ 0.004) and 90.0% (P ¼ 0.001), respectively, but did not have a significant effect on urinary DHBMA. These results reveal a potentially protective effect of PEITC treatment with respect to the detoxification of 1,3-butadiene in cigarette smokers, specifically in those null for GSTT1, and provide further evidence in support of stronger chemopreventive effects from consumption of dietary isothiocyanates in these individuals.