Although an anterior cruciate ligament (ACL) deficiency induces knee osteoarthritis, particularly in the medial compartment, the kinematics remains partially unclear. This study investigated the in vivo knee kinematics of ACL-deficient and normal knees by comparing them during squatting. This prospective comparative study included 17 ACL-deficient knees and 20 normal knees. The kinematics was investigated under fluoroscopy using a two- to three-dimensional registration technique. The anteroposterior (AP) translation of the medial and lateral sides of the femur, axial rotation of the femur relative to the tibia, and kinematic pathways were evaluated and compared. At first, the medial AP position of the femur translated anteriorly from 0° to midflexion, afterward it translated posteriorly in both ACL-deficient and normal knees. However, the medial AP position of the femur in the ACL-deficient knees was located significantly posteriorly compared with normal knees at 0–110° flexion. The lateral AP position of the femur translated posteriorly from 0° to 150° flexion in both ACL-deficient and normal knees. The lateral AP position of the femur in the ACL-deficient knees was located significantly posteriorly compared with that in normal knees at 0–10° flexion. The femur showed external rotation from 0° to 150° flexion in both ACL-deficient and normal knees. A medial pivot motion and subsequent bicondylar rollback were observed in both knees in the kinematic pathway. In conclusion, the AP position of the femur relative to the tibia was altered due to ACL deficiency, particularly in the medial compartment.