Background: Recent evidence supports that restoration of the pre-arthritic condition via total knee arthroplasty (TKA) is associated with improved post-TKA performance and patient satisfaction. However, whether the restored pre-arthritic joint line simulates the native mid-flexion biomechanics remains unclear. Objective: We performed a matched-pair cadaveric study to explore whether restoration of the joint line via kinematically aligned (KA) TKA reproduced native knee biomechanics more accurately than the altered joint line associated with mechanically aligned (MA) TKA. Methods: Sixteen fresh-frozen cadaveric knees (eight pairs) were affixed onto a customized knee-squatting simulator for measurement of femoral rollback and medial collateral ligament (MCL) strain during mid-flexion. One knee from each cadaver was randomly designated to the KA TKA group (with the joint line restored to the pre-arthritic condition) and the other to the MA TKA group (with the joint line perpendicular to the mechanical axis). Optical markers were attached to all knees and rollback was analyzed using motion capture cameras. A video extensometer measured real-time variations in MCL strain. The kinematics and MCL strain prior to and following TKA were measured for all specimens. Results: KA TKA was better for restoring the knee kinematics to the native condition than MA TKA. The mid-flexion femoral rollback and axial rotation after KA TKA were consistently comparable to those of the native knee. Meanwhile, those of MA TKA were similar only at ≤40° of flexion. Furthermore, KA TKA better restored the mid-flexion MCL strain to that of the native knee than MA TKA. Over the entire mid-flexion range, the MCL strain of KA TKA and native knees were similar, while the strains of MA TKA knees were more than twice those of native knees at >20° of flexion. Conclusions: The restored joint line after KA TKA effectively reproduced the native mid-flexion rollback and MCL strain, whereas the altered joint line after MA TKA did not. Our findings may explain why patients who undergo KA TKA experience superior outcomes and more natural knee sensations during daily activities than those treated via MA TKA.