The preservation of the posterior cruciate ligament in cruciate retaining (CR) total knee arthroplasty (TKA) designs has the potential to restore healthy knee biomechanics; however, concerns related to kinematic asymmetries during functional activities still exist in unilateral TKA patients. As there is a limited data available regarding the ability of the contemporary CR TKA design with concave medial and convex lateral tibial polyethylene bearing components to restore healthy knee biomechanics, this study aimed to investigate in vivo three-dimensional knee kinematics in CR TKA patients during strenuous knee flexion activities and gait. Using a combined computer tomography and dual fluoroscopic imaging system approach, in vivo kinematics of 15 unilateral CR TKA patients (comparison of replaced and contralateral nonreplaced knee) were evaluated during sit-to-stand, step-ups, single-leg deep lunge, and level walking. The patient cohort was followed-up at an average of 24.5 months ( ± 12.6, range 13–42) from surgical procedure. Significantly smaller internal knee rotation angles were observed for the contemporary CR TKA design during step-ups (2.6 ± 5.8 vs. 6.3 ± 6.6 degrees, p < 0.05) and gait (0.6 ± 4.6 vs. 6.3 ± 6.8 degrees, p < 0.05). Significantly larger proximal and anterior femoral translations were measured during sit-to-stand (34.7 ± 4.5 vs. 29.9 ± 3.1 mm, p < 0.05; –2.5 ± 2.9 vs. –8.1 ± 4.4 mm, p < 0.05) and step-ups (34.1 ± 4.5 vs. 30.8 ± 2.9 mm, p < 0.05; 2.2 ± 3.2 vs. –3.5 ± 4.5 mm, p < 0.05). Significantly smaller ranges of varus/valgus and internal/external rotation range of motion were observed for CR TKA, when compared with the nonoperated nee, during strenuous activities and gait. The preservation of the posterior cruciate ligament in the contemporary asymmetric bearing geometry CR TKA design with concave medial and convex lateral tibial polyethylene bearing components has the potential to restore healthy knee biomechanics; however, the study findings demonstrate that native knee kinematics were not fully restored in patients with unilateral asymmetric tibial polyethylene bearing geometry CR TKA during functional activities.