Purpose. The purpose of this study was to compare the accuracy and clinical outcomes of the medial open wedge high tibial osteotomy (MOWHTO) using a three-dimensional (3D-) printed patient-specific instrumentation (PSI) with that of conventional surgical techniques. Methods. A prospective comparative study which included 18 patients who underwent MOWHTO using 3D-printed PSI technique (3D-printed group) and 19 patients with conventional technique was conducted from Jan 2019 to Dec 2019. After the preoperative planning, 3D-printed PSI (cutting guide model) was used in MOWHTO for 3D-printed group, while freehand osteotomies were adopted in the conventional group. The accuracy of MOWHTO for each method was compared using the radiological index obtained preoperatively and postoperatively, including mechanical femorotibial angle (mFTA) and medial mechanical proximal tibial angle (mMPTA), and correction error. Regular clinical outcomes were also compared between the 2 groups. Results. The correction errors in the 3D-printed group were significantly lower than the conventional group (mFTA,
0.2
°
±
0.6
°
vs.
1.2
°
±
1.4
°
,
P
=
0.004
) (mMPTA,
0.1
°
±
0.4
°
vs.
2.2
°
±
1.8
°
,
P
<
0.00001
). There was a significantly shorter duration (
P
<
0.00001
) and lower radiation exposures (
P
<
0.00001
) for the osteotomy procedure in the 3D-printed group than in the conventional group. There were significantly higher subjective IKDC scores (
P
=
0.009
) and Lysholm scores (
P
=
0.03
) in the 3D-printed group at the 3-month follow-up, but not significantly different at other time points. Fewer complications occurred in the 3D-printed group. Conclusions. With the assistance of the 3D-printed patient-specific cutting guide model, a safe and feasible MOWHTO can be conducted with superior accuracy than the conventional technique.