Aims: Youkenafil is a novel selective phosphodiesterase type 5 inhibitor to treat erectile dysfunction. Since it was mainly metabolized through cytochrome P450 3A4/5 (CYP3A4/5) in vitro, the effects of itraconazole and rifampicin (potent CYP3A4/5 inhibitor and inducer, respectively) on the pharmacokinetics of youkenafil and its main metabolite (M1) were investigated in two clinical studies. Methods: Each study enrolled thirty healthy male subjects. In study 1, subjects were given a single dose of youkenafil (50 mg on Days 1 and 13) and multiple doses of itraconazole (200 mg once daily from Days 6 to 14). In study 2, subjects were given a single dose of youkenafil (100 mg on Days 1 and 20) and multiple doses of rifampicin (600 mg once daily from Days 6 to 20). Results: Itraconazole significantly increased the systemic exposure to youkenafil and M1. The geometric mean ratios (GMRs) and their 90% confidence intervals (CIs) for the AUC0-t, AUC0-∞ and Cmax of youkenafil were 1198.58% (900.33%–1595.63%), 1162.54% (874.69%–1545.11%) and 632.17% (500.40%–798.64%), respectively. Conversely, rifampicin significantly decreased the systemic exposure to youkenafil but had a slight effect on M1. The GMRs (90% CI) for the AUC0-t, AUC0-∞ and Cmax of youkenafil were 1.72% (1.27%–2.33%), 1.80% (1.33%–2.43%) and 1.77% (1.27%–2.47%), respectively. All subjects tolerated well in both studies. Conclusion: Itraconazole increased youkenafil AUC and Cmax by 12- and 6-fold, respectively. Rifampicin decreased youkenafil AUC and Cmax both by about 98%. Therefore, combined administration of youkenafil with potent inhibitors or inducers of CYP3A4/5 should be avoided or carefully monitored.