Purpose: Although unilateral total knee arthroplasty (TKA) is successful in restoring function, there are concerns in relation to asymmetric gait patterns. Bi-cruciate retaining (BCR) TKA design preserves both anterior and posterior cruciate ligaments with the potential to restore normal joint kinematics. The purpose of this study was to investigate the in vivo three-dimensional (3D) kinematics of the knee in unilateral BCR TKA patients during gait. Methods: Twenty-nine unilateral BCR TKA patients were evaluated for both knees during treadmill gait using a combined computer tomography and dual fluoroscopic imaging system approach. Results: Significantly higher flexion (4 ± 6.7°) and internal rotation (4.3 ± 4.7°) and significantly lower adduction (2.5 ± 4.4°) during the stance phase of gait cycle were observed in the implanted side of unilateral BCR TKA patients. Significant asymmetric lateral/medial (3.2 ± 4.8 mm) and anterior/posterior (3.4 ± 4.1 mm) tibial translations were also measured during treadmill gait in this cohort of patients. Conclusion: Despite maintaining anteroposterior stability, asymmetric knee motion persisted in in vivo unilateral BCR TKA patients during gait. The results of the current study suggested that the knee motion symmetry during gait was not restored in patients with unilateral BCR TKA.