Abstract:[Objective] To compare the clinical consequences of medial open-wedge high tibial osteotomy (MOWHTO) by assistance with 3D printed patient-specific instrument (PSI) guides versus the conventional freehand technique for medial knee osteoarthritis. [Meth- ods] A retrospective study was conducted on 69 patients who received MOWHTO in our department from October 2017 to March 2021. Ac- cording to doctor-patient communication, 37 patients were treated with PSI guides (3D group), while other 32 patients were treated with tradi- tional freehand methods (routine group). The data regarding perioperative period, follow- up and imaging were compared between the two groups. [Results] The 3D group proved significantly superior to the routine group in terms of intraoperative bleeding [(48.7±39.9) ml vs (68.9±50.5) ml, P<0.05], fluoroscopy times [(10.5±1.4) times vs (15.4±2.0) times, P<0.05], despite of insignificant differences in operation time and incision healing grade between the two groups (P>0.05). The mean follow-up time was of (23.6±1.6) months, and there was no sig- nificant difference in time to resume full weight-bearing activity between the two groups (P>0.05). The knee extension-flexion ROM, HSS score, VAS score and WOMAC score significantly improved in both groups over time (P<0.05), whereas which were not significantly differ- ent between the two groups at the last follow-up (P>0.05). In terms of imaging, the K-L classification for osteoarthritis remained unchanged with time in both groups (P>0.05). At any corresponding time point, there were no significant differences in FTA, MPTA and PTS between the two groups (P>0.05). [Conclusion] The application of 3D printed PSI guides does reduce the intraoperative fluoroscopy times and blood loss, but has no significant impact on the operative time of the MOWHTO for medial knee osteoarthritis.