Objective: To analyze and compare the differences between 3D-printed patient-specific instrumentation (PSI)-assisted medial open-wedge high tibial osteotomy (OWHTO) and traditional medial OWHTO in terms of postoperative mechanical stability, accuracy of weight-bearing alignment adjustment, and clinical outcomes. By evaluating the biomechanical performance of the two methods, this study aims to explore the potential advantages of 3D printing technology in improving surgical precision and reducing postoperative complications. Methods: Patients diagnosed with knee osteoarthritis (KOA) and undergoing OWHTO at the First Affiliated Hospital of Soochow University from January 2019 to January 2022 were collected. Patients were divided into the traditional method group (23 individuals) and the 3D-printed PSI-assisted group (18 individuals) based on the surgical methods. The accuracy of correction between the two methods was evaluated by comparing the preoperative planned target correction of the hip-knee-ankle (HKA) angle and the postoperative HKA angle difference. Similarly, the preoperative posterior tibial slope (PTS) and the postoperative PTS angle difference were also assessed. The clinical efficacy of the two methods was assessed by collecting and analyzing the Lysholm score, visual analogue scale (VAS), and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) of the patients in both groups before surgery. This assessment was also conducted at 1, 6, 12, and 24 months postoperatively. The occurrence of postoperative complications in both groups was collected and analyzed to evaluate the safety of PSI-assisted OWHTO. Results: A total of 41 patients were included in this study, with 23 in the traditional method group and 18 in the PSI-assisted group. The demographic characteristics, preoperative imaging, and clinical symptoms were similar between the two groups, showing no statistical difference (P>0.05). Regarding the results of correction accuracy, the postoperative HKA angle difference was 2.7±1.8° in traditional OWHTO group and 0.8±1.1° in 3D-printed PSI-assisted OWHTO group, with a significant difference between the two groups (P<0.001). The postoperative PTS angle difference was 2.8±2.2° for traditional OWHTO and 1.7±1.9° for PSI-assisted OWHTO, showing a significant statistical difference (P=0.003). In terms of clinical efficacy, the surgical time for the PSI-assisted group was 59.2±14.8 minutes, significantly shorter than the traditional method group's 87.6±21.4 minutes (P=0.019). The Lysholm, VAS, and WOMAC scores of the PSI-assisted group were superior to those of the traditional method group at each postoperative follow-up time point. Regarding postoperative complications, there were 4 cases (17.3%) in the traditional method group and 3 cases (16.7%) in the PSI-assisted group, with no significant statistical difference between the two groups. Conclusion: Compared to traditional method, 3D-printed PSI-assisted OWHTO demonstrates superior accuracy in correcting lower limb alignment, along with favorable clinical efficacy and safety. The results of this study provide useful references for clinical doctors in selecting surgical treatment plans.