Aim: The aim of this study was to use bupropion as a cytochrome P450 (CYP) 2B6 probe to determine CYP2B6 function by using a population pharmacokinetic approach of parent bupropion and metabolite hydroxybupropion to explore the impacts of chronic kidney disease (CKD) and vitamin D status in patients and control subjects. Methods: Plasma concentration data from 19 subjects after administration of 150 mg of sustained release bupropion were used in pharmacokinetic modeling. Blood samples were collected pre-dose and serially post-dose and analyzed by LC-MS/MS. Population pharmacokinetic analysis was conducted using non-linear mixed-effect modeling (Phoenix® NLME v8.3, Certara Inc.). A separate post-hoc analysis evaluated the formation clearance of hydroxybupropion as an assessment of CYP 2B6 functional activity. Results: The final model was comprised of 2 compartments with first order formation and elimination for bupropion and hydroxybupropion. A multiplicative error model explained residual variability. Covariates tested in the model, including vitamin D baseline concentrations and CKD, did not have a significant impact on the pharmacokinetics of bupropion and hydroxybupropion. Model validation was performed using visual predictive check. A trend toward increased formation clearance was observed within the CKD group (2.9 ± 1.8 vs. 3.7 ± 2.1, p = 0.43) and the control group (3.5 ± 1.9 vs. 4.1 ± 3.6, p = 0.79) with vitamin D repletion vs. insufficiency. Conclusion: The study contributes to the understanding of CYP 2B6 function in CKD and used a population pharmacokinetic approach to explore the potential impact of vitamin D status on pharmacokinetics of bupropion.