An unintended increase in the posterior tibial slope after open-wedge high tibial osteotomy (HTO) can influence knee kinematics and stability. The objective for this study was to validate the change of the tibial slope obtained from three-dimensional (3D) navigation in open-wedge HTO by comparing it with that evaluated with computed tomography. Human cadaver knees were used. The open-wedge HTO was performed to maintain the anatomic tibial slope according to the navigation system. 3D navigation could provide surgeons with reliable information not only to determine appropriate coronal alignment but also to maintain the anatomic tibial slope in open-wedge HTO.