ABSTRACT Efavirenz's effects on cytochrome P450 2C9 (CYP2C9) activity have not been formally characterized in vivo. We conducted the first clinical drug–drug interaction (DDI) study to test the effect of chronic efavirenz dosing on CYP2C9 activity, using tolbutamide as a selective probe. Healthy participants received a single oral dose of tolbutamide (250 mg) with a single 600 mg dose of efavirenz (SD) before and after 600 mg daily efavirenz for 17 days (MD). Tolbutamide, efavirenz, and their metabolites were quantified in plasma and urine samples by LC–MS/MS method. Participants were genotyped for CYP2C9*2 and *3 and for CYP2B6*4, *9, and *18. Pharmacokinetic data were valid for 71 and 59 participants for SD‐ and MD groups, respectively. MD efavirenz caused more than 50% increase in median tolbutamide AUC 0‐∞ and C 24 in all subjects, and 1.42–1.46‐fold increase in the paired analysis ( p < 0.0001); tolbutamide/metabolite ratios were also increased ( p < 0.001), while tolbutamide's CL/F/kg, metabolite formation clearance, and metabolite C max were significantly reduced. CYP2C9 genetic variants were associated with reduced tolbutamide elimination compared to *1/*1 and *1/*2 (slowest, *3/*3; and intermediate, *1/*3, *2/*2, and *2/*3). The lowest percent change occurred in *2/*2 and * 3/*3 genotypes, though small sample sizes limited reliable assessment of genotype‐specific DDI effects. In conclusion, chronic efavirenz use inhibits CYP2C9 activity in vivo. This inhibition may increase the risk of adverse effects from narrow‐therapeutic‐index CYP2C9 substrates (e.g., warfarin, phenytoin, and sulfonylureas). Therefore, therapeutic drug monitoring and dose adjustments are warranted when efavirenz is co‐administered with these medications.