Despite the efficacy of bupropion for smoking cessation, smokers exhibit variability in treatment outcome. The CYP2B6 gene has been implicated in bupropion kinetics and nicotine metabolism, and is a plausible candidate for pharmacogenetic studies of treatment response. We investigated whether a functional genetic polymorphism in the CYP2B6 gene predicts smoking outcomes in a placebo-controlled randomized trial. Four hundred and twenty-six smokers of European Caucasian ancestry provided blood samples and received bupropion (300 mg/day for 10 weeks) or placebo, plus counseling. Smoking status, abstinence symptoms and side-effects were recorded weekly, and smoking status was verified at the end of treatment and at 6-month follow-up. Smokers with a decreased activity variant of CYP2B6 reported greater increases in cravings for cigarettes following the target quit date and had higher relapse rates. These effects were modified by a significant gender x genotype x treatment interaction, suggesting that bupropion attenuated the effects of genotype among female smokers. We conclude that smokers with the CYP2B6 variant may be more vulnerable to abstinence symptoms and relapse. Bupropion may attenuate these effects, especially among females. Additional trials are warranted to confirm these results, as are studies to explore the neurobiological mechanisms. Such research could ultimately enable practitioners to select the optimal type and dose of medication for individual smokers.