Purpose The purpose of this study is to investigate the effect of sildenafil a CYP3A4 substrate and inhibitor on the pharmacokinetics and safety of saxagliptin. Methods Eighteen healthy volunteers were recruited in sequential; single-center study to determine pharmacokinetic parameters of saxagliptin and sildenafil, and (AUC0-∞), (AUC0-t); Cmax; tmax; t½, ke; ka were measured using validated LC–MS/MS method. Therapeutic doses were given as follows: Sildenafil 50 mg single dose on day one, then washout period from day two till day eight. Saxagliptin 5 mg once/day was given from day 9 till day 12; then on day 13, the two drugs were co-administered. Blood samples for pharmacokinetic analysis were collected on days 1 and 13 for sildenafil and on days 12 and 13 for saxagliptin. Results Saxagliptin ratios of T/R and 90% CI were 132.1% (122.7–142.3) for AUC0–t, and 167.6% (154.6–181.8) for Cmax. On the other hand, sildenafil pharmacokinetics were not affected. Gmax changed from 93.7 mg/dl to 95.6 mg/dl (P > 0.001) and AUCg0-t from 512.8 ng.h/ml to 532.75 ng.h/ml (P > 0.001) after co-administration of both drugs. Conclusion Sildenafil significantly affected the pharmacokinetic parameters of saxagliptin when co-administered. Registration This trial was registered at clinicaltrials.gov under identifier number: [NCT04170790] in November 2019.