Internal tibial rotation is a risk factor for anterior cruciate ligament (ACL) injury. The effect of restraining tibial rotation (RTR) to prevent ACL injury during single-leg landing is not well understood. We aimed to investigate the effect of impact load and RTR on ACL injury with respect to flexion angle. We hypothesized that RTR could protect the knee from ACL injury compared to free tibial rotation (FTR) regardless of flexion angle and create a safety zone to protect the ACL. Thirty porcine specimens were potted in a rig manufactured to replicate single-leg landing maneuvers. A mechanical testing machine was used to apply external forces in the direction of the tibial long axis. A 3D displacement sensor measured anterior tibial translation (ATT). The specimens were divided into 3 groups of 10 specimens and tested at flexion angles of 22 ± 1°, 37 ± 1° and 52 ± 1° (five RTR and five FTR) through a consecutive range of actuator displacements until ACL failure. After dissection, damage to the joint was visually recorded. Two-way ANOVA were utilized in order to compare compressive forces, torques and A/P displacements with respect to flexion angle. The largest difference between peak axial compressive forces (~3.4 kN) causing ACL injury between RTR and FTR was reported at a flexion angle of 22°. Tibial torques with RTR was in the same range and < 20 Nm at the instance and just before ACL failure, compared to a significant reduction when cartilage/bone damage (no ACL failure) was reported. Isolated ACL injuries were observed in ten of the 15 FTR specimens. Injuries to bone and cartilage were more common with RTR. RTR increases the threshold for ACL injury by elevating the compressive impact load required at lower flexion angles. These findings may contribute to neuromuscular training programs or brace designs used to avoid excessive internal/external tibial rotation. Caution must be exercised as bone/cartilage damage may result.