This is an experimental study. Gender has been reported to influence outcomes in patients with total knee arthroplasty (TKA) for knee osteoarthritis (OA). However, the influence of gender on three-dimensional (3D) in vivo kinematics during gait remains unclear. This study aimed to determine if 3D gait kinematics, including 3D knee translations and rotations, differed in men and women following bicruciate-retaining (BCR) TKA. Twenty-nine well-functioning unilateral BCR TKA patients (14 males and 15 females) underwent evaluation of both knees during level walking on a treadmill at a self-selected speed using a dual fluoroscopic imaging system. Interlimb comparisons of in vivo 6 degree-of-freedom kinematics were compared between male and female patients. Differences of pre- A nd postoperative Knee Society scores (KSSs) were compared between the groups. Both groups were matched regarding age and body mass index. Both male and female patients demonstrated improvement in their postoperative KSSs. Statistically significant differences were observed with respect to spatiotemporal anterior-posterior interlimb translations (p < 0.05). Although females presented more femoral posterior translation in the operative knee than the nonoperative knee during most of the stance phases (2.8 vs.-1.6 mm), males exhibited less femoral translation in the operative knee than the nonoperative knee (2.3 vs.-1.8 mm), when interlimb differences were detected during stance phase. Results demonstrated that there are 3D motion asymmetries of the knee in both male and female unilateral BCR TKA patients during gait with anterior-posterior interlimb asymmetries significantly greater in female than male participants. This suggests that gender may influence the in vivo knee kinematics in BCR TKA patients during gait.