To evaluate the influence of tibial and femoral tunnel position in ACL reconstruction on knee kinematics, we compared ACL reconstruction with a tibial and femoral tunnel in anteromedial (AM-AM reconstruction) and in posterolateral footprint (PL-PL reconstruction) with a reconstruction technique with tibial posterolateral and femoral anteromedial tunnel placement (PL-AM reconstruction). In 9 fresh-frozen human cadaveric knees, the knee kinematics under simulated Lachman (134 N anterior tibial load) and a simulated pivot shift test (10 N/m valgus and 4 N/m internal tibial torque) were determined at 0°, 30°, 60°, and 90° of flexion. Kinematics were recorded for intact, ACL-deficient, and single-bundle ACL reconstructed knees using three different reconstruction strategies in randomized order: (1) PL-AM, (2) AM-AM and (3) PL-PL reconstructions. Under simulated Lachman test, single-bundle PL-AM reconstruction and PL-PL reconstructions both showed significantly increased anterior tibial translation (ATT) at 60° and 90° when compared to the intact knee. At all flexion angles, AM-AM reconstruction did not show any statistical significant differences in ATT compared to the intact knee. Under simulated pivot shift, PL-AM reconstruction resulted in significantly higher ATT at 0°, 30°, and 60° knee flexion and AM-AM reconstructions showed significantly higher ATT at 30° compared to the intact knee. PL-PL reconstructions did not show any significant differences to the intact knee. AM-AM reconstructions restore the intact knee kinematics more closely when compared to a PL-AM technique resembling a transtibial approach. PL-PL reconstructions showed increased ATT at higher flexion angles, however, secured the rotational stability at all flexion angles. Due to the independent tibial and femoral tunnel location, a medial portal technique may be superior to a transtibial approach.