Background and purpose: There is high interindividual variability in the activity of drug‐metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug–drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored.Experimental approach: A randomized crossover double‐blind placebo‐controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg·kg−1and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session.Key results: CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and Cmax(−0.71 < Spearman correlation coefficient ρs < −0.92). Oxymorphone Cmaxwas 62% and 75% lower in PM than EM and UM. Noroxymorphone Cmaxreduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone Cmaxby 40% and 80%, and increased noroxycodone AUC∞by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC∞and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition.Conclusions and implications: Drug–drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype.