Lemborexant is a novel dual orexin receptor antagonist under development for the treatment of primary insomnia and irregular sleep-wake rhythm disorder. In vitro assessments using recombinant enzymes demonstrated that CYP3A is mainly involved in lemborexant metabolism. The objectives of the present study were to investigate the effect of itraconazole, a strong CYP3A inhibitor, on the pharmacokinetic (PK) profiles of lemborexant in healthy volunteers and to simulate drug-drug interactions (DDIs) with weak and moderate CYP3A inhibitors using physiologically-based pharmacokinetic (PBPK) modeling. Fifteen subjects received single oral doses of 10 mg lemborexant once prior to and once during 20 days of daily dosing with 200 mg itraconazole to characterize the effect of itraconazole on PK profiles of lemborexant. The coadministration of itraconazole resulted in increases of 1.36-fold and up to 3.70-fold in Cmax and AUC of lemborexant, respectively. These results demonstrated that CYP3A contributes mainly to lemborexant metabolism in humans. A PBPK model of lemborexant was developed using the Simcyp population-based simulator (v14r1) with in vitro data on plasma protein binding, information on physicochemical properties, and human oral clearance data. The PK profiles observed in combination with itraconazole were also compared with simulated results and used for the model verification. For DDI simulations, default library compound files for CYP3A inhibitors (itraconazole, diltiazem, erythromycin, fluconazole, verapamil, fluoxetine, and fluvoxamine) were used. The developed PBPK model closely predicted the observed PK profiles of lemborexant after a single oral dose of lemborexant alone and in combination with itraconazole. The predicted PK parameters were comparable to observed results, supporting verification of the PBPK model. DDI simulations using the developed PBPK model were conducted with the same dosing schedule as itraconazole to assess impacts of moderate and weak CYP3A inhibitors on PK profiles of lemborexant. Effects of moderate inhibitors on lemborexant were mild to moderate as demonstrated by a 1.25 to 1.39-fold and 2.02 to 3.91-fold increases in Cmax and AUC of lemborexant, respectively. The weak inhibitors did not affect the PK profiles of lemborexant. These simulation results support coadministration of lemborexant and weak CYP3A inhibitors without any dose adjustment of lemborexant. Further evaluations of impacts of moderate CYP3A inhibitors are needed to fully understand appropriate use of lemborexant with moderate CYP3A inhibitors in clinical situations.