Nivasorexant, a selective orexin‐1‐receptor antagonist, has recently been assessed in the treatment of humans with binge‐eating disorder. Herein, the inhibitory potential of nivasorexant on cytochromes P450 (CYPs) 2C9, 2C19, and 3A4 was evaluated. Human liver microsomes/recombinant CYP enzymes were evaluated in vitro. In vivo, a single‐center, open‐label, fixed‐sequence study was performed in healthy adults to explore the effect of 100 mg nivasorexant administered twice daily (b.i.d.) on the pharmacokinetics (PK) of flurbiprofen (50 mg, CYP2C9), omeprazole (20 mg, CYP2C19), midazolam (2 mg, CYP3A4) making use of a cocktail approach. Plasma PK sampling was performed over 24 h on Day 1 (Cocktail alone), 8 (Cocktail + nivasorexant), and 15 (Cocktail + nivasorexant at steady state). Genotyping of subjects' CYPs was performed while safety and tolerability were also assessed. In vitro, nivasorexant inhibited CYP2C9, 2C19, and 3A4 in competitive inhibition assays with IC50 values of 8.6, 1.6, and 19–44 μM, respectively, while showing a significant time‐dependent CYP2C19 inhibition. In 22 subjects, exposure to flurbiprofen, omeprazole, and midazolam was generally higher during concomitant single‐ (i.e., area under the plasma concentration–time curve [AUC] ratio increased by 1.04‐, 2.05‐, and 1.56‐fold, respectively) and repeated‐dose (i.e., AUC ratio increased by 1.47‐, 6.84‐, and 3.71‐fold, respectively) nivasorexant administration compared with the cocktail substrates administered alone. The most frequently reported adverse event was somnolence. According to regulatory guidance, nivasorexant is classified as a moderate CYP2C19 and weak CYP3A4 inhibitor after 1 day and as a weak CYP2C9, strong CYP2C19, and moderate CYP3A4 inhibitor after 8 days of 100 mg b.i.d. administration. Clinicaltrials.gov ID: NCT05254548.