Objective To investigate the effect of medial unicompartmental knee arthroplasty (UKA) surgery on knee biomechanics during stair ascent. Methods Nine osteoarthritis patients who received fixed-bearing medial UKA participated in this study. All patients completed pre-surgical (3 weeks before UKA surgery) and post-surgical ［(7±2) months after UKA surgery］ test. Their synchronized biplane radiographs during stair ascent were collected. Motion of the femur, tibia, and implants were tracked using an automated volumetric model-based tracking process that matched subject-specific 3D models of the bones and prostheses to the biplane radiographs with sub-millimeter accuracy. Anatomic coordinate systems were created within the femur and tibia and used to calculate tibiofemoral kinematics. Additional outcome measures included the center of contact in the medial and lateral compartments, and the lateral compartment dynamic joint space. Results The UKA knee was in 4.8° varus compared with the pre-surgical contralateral knee. The post-surgical UKA knee was in 3.1°valgus compared with the pre-surgical knee. The post-surgical UKA knee was 4.4° externally rotated compared with the pre-surgical contralateral knee. However, the medial tibia contact center of the UKA knee moved posteriorly 2.5 mm compared with that of the contralateral knee (P<0.05). No obvious changes were found in lateral compartment joint space before and after surgery (P>0.05). Conclusions UKA can effectively improve varus of the knee joint and restore biomechanical characteristics of UKA knee rotation, without affecting lateral compartment joint space. However, changes are found in contact center of the medial tibia compartment of the UKA knee after surgery.