Background Physiological tibiofemoral kinematics have been shown to be important for good knee function after total knee arthroplasty (TKA). The purpose of the present study was to investigate the influence of component rotation on tibiofemoral kinematics during knee flexion. We asked which axial component alignment best reconstructs physiological tibiofemoral kinematics and which combinations should be avoided. Methods Ten healthy cadaveric knees were examined. By means of a navigational device, tibiofemoral kinematics between 0° and 90° of flexion were assessed before and after TKA using the following different rotational component alignment: femoral components: ligament balanced, 6° internal, 3° external rotation, and 6° external rotation in relation to the posterior condylar line; tibial components: self-adapted, 6° internal rotation, and 6° external rotation. Results Physiological tibiofemoral kinematics could be partly reconstructed by TKA. Ligament-balanced femoral rotation and 6° femoral external rotation both in combination with 6° tibial component external rotation, and 3° femoral external rotation in combination with 6° tibial component internal rotation or self-aligning tibial component were able to restore tibial longitudinal rotation. Largest kinematical differences were found for the combination femoral component internal and tibial component external rotations. Conclusion From a kinematic-based view, surgeons should avoid internal rotation of femoral components. However, even often recommended combinations of rotational component alignment (3° femoral external and tibial external rotation) significantly change tibiofemoral kinematics. Self-aligning tibial components solely restored tibiofemoral kinematics with the combination of 3° femoral component of external rotation. For the future, navigational devices might help to axially align components to restore patient-specific and natural tibiofemoral kinematics.