Background: Once-daily tacrolimus administration has demonstrated improved medication compliance in compared to twice-daily dosing. However, the safety and efficacy of generic once-daily tacrolimus in de novo kidney transplant recipients remain underexplored. Methods: We investigated the efficacy and safety of generic once-daily tacrolimus during the 6 months following de novo kidney transplantation. This prospective, multicenter, open-label, single-arm study was conducted across 10 transplant centers (NCT03749356). The primary endpoint was the composite efficacy failure rate, including biopsy-proven acute T-cell mediated rejection, graft loss, death or loss to follow-up, within 24 weeks posttransplantation. Among the 147 screened kidney transplant candidates, 141 were enrolled, and 121 successfully completed the study. Results: The primary efficacy failure rates were 5.0% in the full-analysis set and 5.8% in the per-protocol set. During the follow-up, there were no cases of graft loss, but one patient died with a functioning graft due to acute respiratory distress syndrome. Eight patients experienced biopsy-proven acute rejection (six T-cell mediated rejections and two antibody-mediated rejections). The mean estimated glomerular filtration rate at 24 weeks was 64.7 mL/min/1.73 m2. In the full-analysis set, adverse events and serious adverse events with a suspected relation to study drug occurred in 56.0% and 8.5% of cases, respectively. Of the 17 participants who dropped out from the study, 7 discontinued study drug due to difficulties in regulating whole-blood tacrolimus trough concentrations within the target range. Conclusions: This study suggests that generic once-daily tacrolimus is both effective and safe for use in de novo kidney transplant patients.