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Background: Opening wedge high tibial osteotomy (OWHTO) is an effective surgical treatment for knee osteoarthritis. This study aimed to explore the feasibility and accuracy of a novel 3D-printed patient-specific instrument (PSI) based on "H-point" for medial OWHTO in a prospective cadaver study. Methods: Twenty-six fresh-frozen lower limbs were collected and randomly divided into two groups: PSI group treated with 3D virtual preoperative planning and a novel 3D-printed PSI; control group with the standard technique. 3D models were reversely reconstructed for preoperative surgical planning, guide plate design, and simulated osteotomy. Anatomic features of "H-point," surgical time, fluoroscopic dose, correction accuracy including tibiofemoral angle (FTA) and posterior tibial slope (TS) angle were measured. Results: First, H-point was always described as a bony bulge in the posteromedial to the proximal tibia and had a relatively constant relationship with the osteotomy site. Second, the absolute correction error of mFTA and TS were significantly smaller in the PSI group. The effective rate of TS in the PSI group was more concentrated with absolute correction error within 1° and within 2° for 53.3% and 93.3%, compared to 9.1% and 45.5% in the control group. The total operation time, positioning osteotomy time, distraction correction time and fluoroscopy dose in the PSI group were significantly less than those in the control group. Conclusions: The novel 3D-printed PSI based on H-point is feasibility and accuracy with advantages in terms of TS, surgery time and radiation dose for OWHTO. [ABSTRACT FROM AUTHOR]