BACKGROUND: Kinematic alignment (KA) unicompartmental knee arthroplasty (UKA), which has not been widely adopted in clinical practice, aims to implant a more personalized and physiologically compatible mobile-bearing UKA prosthesis for the treatment of advanced single compartment knee osteoarthritis. KA UKA is anticipated to enhance patient satisfaction and decrease the revision rate following UKA. However, its quantified biomechanical indicators remain unclear. The purpose of this study is to reveal the biomechanical characteristics of the tibiofemoral joint in normal and KA UKA knees, and to evaluate the biomechanical effect. METHODS: In this study, six cadaveric knee joint specimens were utilized for biomechanical testing before (normal cadaveric knee joint specimen ) and after KA UKA. The knee joint specimens were subjected to an axial load of 1000 N, and the biomechanical parameters were assessed at flexion angles ranging from 0° to 120° in 10° increments. RESULTS: The root mean square (RMS) values of the tibiofemoral contact area, mean contact pressure, and peak contact pressure during knee flexion were 529 mm², 1.8 MPa, and 4.5 MPa in normal knees, respectively. After KA UKA, these values changed to 449 mm², 2.0 MPa, and 9.8 MPa, respectively. Additionally, the RMS value of the external rotation of the femur relative to the tibia in the tibiofemoral joint was 9.9° in normal knees, while the posterior translations of the center of the femoral condyle, the medial femoral condyle, and the lateral femoral condyle were 18.4 mm, 11.5 mm, and 25.4 mm respectively. After KA UKA, these values changed to 8.6°, 19.3 mm, 12.9 mm, and 25.9 mm respectively. CONCLUSION: At the same flexion angle, the increase in peak contact pressure in the medial compartment after KA UKA is the most significant compared with the normal knees. However, the kinematic characteristics do not change significantly after KA UKA. These findings are beneficial for understanding the possible postoperative complications and good functional effects of KA UKA.