Metabolism of nicotine, the main psychoactive compound in cigarettes, increases in pregnancy which may explain why pregnant women find it difficult to quit smoking and why nicotine replacement therapies have failed in pregnancy. Our study aimed to determine the extent and timing of changes in nicotine metabolic pathways, including C-oxidation and N-glucuronidation, and the pregnancy-induced influences on the activity of enzymes mediating these pathways (CYP2A6 and UGT2B10, respectively). Current smoking pregnant women (n=47) provided a urine sample at early pregnancy (12 weeks), late pregnancy (28 weeks), and 6 months postpartum. Concentrations of urinary nicotine and all metabolites (total nicotine equivalents, TNE) were analyzed using LCMS/MS. General linear repeated measures analyses with Bonferroni correction for multiple testing were used to compare the fraction of nicotine metabolites and metabolic pathways between pregnancy stages. Nicotine C-oxidation was 7% and 11% higher at early and late pregnancy, respectively, compared to postpartum. Nicotine N-glucuronidation was 33% and 67% higher at early and late pregnancy, respectively, compared to postpartum. The CYP2A6 metabolite phenotype ratio (total 3-hydroxycotinine/cotinine) was 56% and 100% higher at early and late pregnancy, respectively, compared to postpartum. This ratio correlated significantly with nicotine C-oxidation suggesting CYP2A6 activity, responsible for the first step in nicotine C-oxidation, may be induced during pregnancy. Similarly, the UGT2B10 metabolite phenotype ratio (nicotine glucuronide/nicotine) was 113% and 322% higher at early and late pregnancy, respectively, compared to postpartum. This ratio correlated significantly with a second UGT2B10 metabolite ratio (cotinine glucuronide/cotinine) suggesting UGT2B10 activity, responsible for nicotine and its primary metabolite cotinine’s glucuronidation, may be induced during pregnancy. In conclusion, pregnancy-induced increases in nicotine C-oxidation and N-glucuronidation occur as early as 12 weeks gestation and increase further as pregnancy progresses compared to non-pregnant levels at 6 months following birth. The substantial increase in nicotine’s metabolism is likely due to induction of CYP2A6 and UGT2B10 activity. These findings have implications for metabolism of other clinically relevant substrates of CYP2A6 and UGT2B10 during pregnancy.