Anterior cruciate ligament (ACL) deficiency increases the risk of early osteoarthritis (OA). Studies of ACL deficient knee kinematics would be important to reveal the disease process and therefore to find mechanisms which would potentially slow OA progression. The purpose of this study was to determine if in vivo kinematics of the anterior cruciate ligament deficient (ACLD) knee during a wide-based squat activity differ from kinematics of the contralateral intact knee. Thirty-three patients with a unilateral ACLD knee consented to participate in this institutional review board approved study with the contralateral intact knee serving as the control. In vivo knee kinematics during the wide-based squat were analyzed using a 2D/3D registration technique utilizing CT-based bone models and lateral fluoroscopy. Comparisons were performed using values between 0 and 100° flexion both in flexion and extension phases of the squat activity. Both the ACLD and intact knees demonstrated increasing tibial internal rotation with knee flexion, and no difference was observed in tibial rotation between the groups. The tibia in the ACLD knee was more anterior than that of the contralateral knees at 0 and 5° flexion in both phases (p < 0.05). Tibiofemoral medial contact points of the ACLD knees were more posterior than that of the contralateral knees at 5, 10 and 15° of knee flexion in the extension phase of the squat activity (p < 0.05). Tibiofemoral lateral contact points of the ACLD knees were more posterior than that of the contralateral knees at 0° flexion in the both phases (p < 0.05). The kinematics of the ACLD and contralateral intact knees were similar during the wide-based squat except at the low flexion angles. Therefore, we conclude the wide-based squat may be recommended for the ACLD knee by avoiding terminal extension.