Purpose The role of the anterior cruciate ligament (ACL) in knee biomechanics in vivo and under weight-bearing is still unclear. The purpose of this study was to compare the tibiofemoral kinematics of ACL-deficient knees to healthy contralateral ones during the execution of weight-bearing activities. Methods Eight patients with isolated ACL injury and healthy contralateral knees were included in the study. Patients were asked to perform a single step forward and a single leg squat first with the injured knee and then with the contralateral one. Knee motion was determined using a validated model-based tracking process that matched subject-specific MRI bone models to dynamic biplane radiographic images, under the principles of Roentgen stereophotogrammetric analysis (RSA). Data processing was performed in a specific software developed in Matlab. Results Statistically significant differences ( p  < 0.05) were found for single leg squat along the frontal plane: ACL-deficient knees showed a more varus angle, especially at the highest knee flexion angles (40°–50° on average), compared to the contralateral knees. Furthermore, ACL-deficient knees showed tibial medialization along the entire task, while contralateral knees were always laterally aligned. This difference became statistically relevant ( p  < 0.05) for knee flexion angles included between 0° and about 30°. Conclusion ACL-deficient knees showed an abnormal tibial medialization and increased varus angle during single leg squat when compared to the contralateral knees. These biomechanical anomalies could cause a different force distribution on tibial plateau, explaining the higher risk of early osteoarthritis in ACL deficiency. The clinical relevance of this study is that also safe activities used in ACL rehabilitation protocols are significantly altered in ACL deficiency. Level of evidence III.