High tibial osteotomy is becoming increasingly popular but can be associated with unintentional posterior tibial slope (PTS) increase and subsequent anterior cruciate ligament (ACL) degeneration. This study quantified the effect of increasing PTS on knee kinematics and in situ forces in the native ACL. A robotic testing system was used to apply external loads from full extension to 90° flexion to seven human cadaveric knees:​ (1) 200 N axial compressive load, (2) 5 Nm internal tibial + 10 Nm valgus torque, and (3) 5 Nm external tibial + 10 Nm varus torque. Kinematics and in situ forces in the ACL were acquired for the native and increased PTS state. Increasing PTS resulted in increased anterior tibial translation at 30° (1.8 mm), 60° (1.7 mm), and 90° (0.9 mm) flexion and reduced in situ force in the ACL at 30° (57.6%), 60° (69.8%), and 90° (75.0%) flexion in response to 200 N axial compressive load. In response to 5 Nm internal tibial + 10 Nm valgus torque, there was significantly less (39.0%) in situ force in the ACL at 90° flexion in the increased compared with the native PTS state. Significantly less in situ force in the ACL at 60° (62.8%) and 90° (67.0%) flexion was observed in the increased compared with the native PTS state in response to 5 Nm external tibial + 10 Nm varus torque. Increasing PTS affects knee kinematics and results in a reduction of in situ forces in the native ACL during compressive and rotatory loads at flexion angles exceeding 30°. In a controlled laboratory setting PTS increase unloads the ACL, affecting its natural function.