Cytochrome P450 (CYP) 3A5 polymorphism influences tacrolimus metabolism, but its effect on the drug pharmacokinetics in liver transplant recipients switched to once-daily extended-release formulation remains unknown. The aim of this study is to analyze the effect of CYP3A5 polymorphism on liver function after once-daily tacrolimus conversion in liver transplant patients. A prospective open-label study included 60 stable liver transplant recipients who underwent 1:1 conversion from twice-daily tacrolimus to once-daily tacrolimus. All participants were genotyped for CYP3A5 polymorphism. The study was registered at ClinicalTrials.gov (NCT 02882113). Twenty-eight patients were enrolled in the CYP3A5 expressor group and 32 in the non-expressor group. Although there was no statistical difference, incidence of liver dysfunction was higher in the expressor group than in the non-expressor group when converted to once-daily extended-release tacrolimus (p = 0.088). No biopsy-proven acute rejection, graft failure, and mortality were observed in either group. The decrease in dose-adjusted trough level (−42.9% vs. −26.1%) and dose/kg-adjusted trough level of tacrolimus (−40.0% vs. −23.7%) was significantly greater in the expressor group than in the non-expressors after the conversion. A pharmacokinetic analysis was performed in 10 patients and tacrolimus absorption in the non-expressor group was slower than in the expressor group. In line with this observation, the area under the curve for once-daily tacrolimus correlated with trough level (Cmin) in the non-expressors and peak concentration (Cmax) in the expressors. CYP3A5 genotyping in liver transplant recipients leads to prediction of pharmacokinetics after switching from a twice-daily regimen to a once-daily dosage form, which makes it possible to establish an appropriate dose of tacrolimus.