Background: Tobacco exposure is routinely assessed by quantifying nicotine metabolites in plasma or urine. On average, 80% of nicotine undergoes C-oxidation to cotinine. However, interindividual variation in nicotine glucuronidation is substantial, and glucuronidation accounts for from 0% to 40% of total nicotine metabolism. We report here the effect of a polymorphism in a UDP-glucuronsyltransferase, UGT2B10, on nicotine metabolism and consumption. Methods: Nicotine, cotinine, their N-glucuronide conjugates, and total trans-3′-hydroxycotinine were quantified in the urine (n = 327) and plasma (n = 115) of smokers. Urinary nicotine N-oxide was quantified in 105 smokers. Nicotine equivalents, the sum of nicotine and all major metabolites, were calculated for each smoker. The relationship of the UGT2B10 Asp67Tyr allele to nicotine equivalents, N-glucuronidation, and C-oxidation was determined. Results: Individuals heterozygous for the Asp67Tyr allele excreted less nicotine or cotinine as their glucuronide conjugates than did wild-type, resulting in a 60% lower ratio of cotinine glucuronide to cotinine, a 50% lower ratio of nicotine glucuronide to nicotine, and increased cotinine and trans-3′-hydroxycotinine. Nicotine equivalents, a robust biomarker of nicotine intake, were lower among Asp67Tyr heterozygotes compared with individuals without this allele: 58.2 (95% confidence interval, 48.9-68.2) versus 69.2 nmol/mL (95% confidence interval, 64.3-74.5). Conclusions: Individuals heterozygous for UGT2B10 Asp67Tyr consume less nicotine than do wild-type smokers. This striking observation suggests that variations in nicotine N-glucuronidation, as reported for nicotine C-oxidation, may influence smoking behavior. Impact: UGT2B10 genotype influences nicotine metabolism and should be taken into account when characterizing the role of nicotine metabolism on smoking.