Tacrolimus is metabolized by cytochrome P450 (CYP) 3A5. The objective of this study was to investigate the influence of the genetic polymorphism of CYP3A5 on the pharmacokinetics of a new modified-release, once-daily formulation of tacrolimus (Advagraf®) after a switch from the immediate-release formulation (Prograf®). This was a prospective, single-centre, open-label study in stable kidney transplant recipients. Seventeen 'expressor' patients (CYP3A5*1/*3 or *1/*1) were matched to 15 'non-expressor' patients (CYP3A5*3/*3). Exposure variables (concentrations and area under the blood concentration-time curve from 0 to 24 hours AUC(24)) were obtained before and 15 days after the switch. Delay since grafting was similar for both groups of patients (expressors: 49 ± 24 months; non-expressors: 45 ± 22 months). During administration of tacrolimus as Prograf® or Advagraf®, the mean tacrolimus daily dose was significantly higher and the dose-adjusted AUC(24) was significantly lower in the expressor group. Following the switch to Advagraf®, there was a significant decrease in the dose-adjusted AUC(24) for both non-expressor (5910 ± 3019 vs 5334 ± 2668 ng·h/mL per mg/kg/day; p = 0.041) and expressor patients (3701 ± 1409 vs 3273 ± 1372 ng·h/mL per mg/kg/day; p = 0.03). In the non-expressor group, mean blood trough concentration (C(0)) was comparable for both formulations while it decreased significantly in the expressor group after the switch (8.2 ± 2.2 vs 6.3 ± 2.5 ng/mL; p = 0.02). However, a good correlation between AUC(24) and C(0) was observed for both Advagraf® and Prograf® regardless of CYP3A5 genotype. Tacrolimus exposure significantly decreases after a switch from Prograf® to Advagraf®, on a milligram-for-milligram basis, in CYP3A5 expressor recipients. Consequently, these patients should be carefully monitored.