Objective To analyze and compare the differences between 3D-printed patient-specific instrumentation (PSI)-assisted medial open-wedge high tibial osteotomy (OWHTO) and conventional medial OWHTO in terms of the postoperative mechanical stability, accuracy of weight-bearing alignment adjustment, and clinical outcomes. Methods: Data from patients diagnosed with knee osteoarthritis (KOA) and undergoing OWHTO from Jan. 2019 to Jan. 2022 were collected. The patients were divided into the conventional method group (23 individuals) and 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 preoperatively planned target correction of the hip–knee–ankle (HKA) angle with the postoperative HKA angle difference. The preoperative posterior tibial slope (PTS) and postoperative PTS angle differences were also assessed. The clinical efficacy of the two methods was assessed by collecting and analyzing the Lysholm score, visual analog scale (VAS), and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) of the patients in both the groups prior to surgery and at the 1st, 6th, 12th, and 24th month postoperatively. The occurrence of postoperative complications in both the groups was analyzed to evaluate the safety of PSI-assisted OWHTO. Results: The demographic characteristics, preoperative imaging observations, and clinical symptoms were similar between the two groups (P>0.05). With regard to the results of correction accuracy, the postoperative HKA angle difference was 2.7°±1.8° in the conventional OWHTO group and 0.8°±1.1° in the 3D-printed PSI-assisted OWHTO group (P<0.001). The postoperative PTS angle difference was 2.8°±2.2° for conventional OWHTO and 1.7°±1.9° for PSI-assisted OWHTO (P=0.003). In terms of clinical efficacy, the surgical time of the PSI-assisted group was 59.2±14.8 min. This was significantly shorter than that of the conventional method group (87.6±21.4 min) (P=0.019). The Lysholm, VAS, and WOMAC scores of the PSI-assisted group were superior to those of the conventional method group at each postoperative follow-up visit. With regard to postoperative complications, there were four cases (17.3%) in the conventional method group and three (16.7%) in the PSI-assisted group. The statistical difference between the two groups is not significant. Conclusions Compared with the conventional method, 3D-printed PSI-assisted OWHTO demonstrated superior accuracy in correcting lower limb alignment, in conjunction with favorable clinical efficacy and safety. This study has provided an effective reference for clinicians in selecting surgical treatment plans.