The pharmacokinetics, efficacy and safety of once-daily tacrolimus formulation (Tac-OD) were assessed in 34 stable pediatric kidney transplant recipients. Enrolled patients received their dose of twice-daily tacrolimus formulation (Tac-BID) on study Days 0 through 7. On the morning of study Day 8, the total daily doses for patients were converted to Tac-OD on a 1:1 basis and maintained on a once-daily morning dosing regimen. Tacrolimus pharmacokinetic profiles were obtained on study Days 7, 14 and 28 (after dose adjustment). Although the mean C0 concentrations (4.10 ± 1.16–3.53 ± 1.10 ng/mL, p = 0.004), and AUC0–24 (151.8 ± 41.6–129.8 ± 39.3 ng h/mL, p < 0.001) were decreased significantly after a 1:1 based conversion, there was high interindividual variability. The dose of Tac-OD was decreased in 26.5% and increased in 44.1% of patients. The resultant tacrolimus dose and pharmacokinetic profiles on study Day 28 were comparable to those on Day 7. There were no serious adverse events. In conclusion, Tac-BID can be safely converted to Tac-OD in stable pediatric kidney transplant patients with the heightened therapeutic drug monitoring. Effects of drug conversion on the cardiovascular risk factors, neurological side effects and adherence should be further evaluated.