Knee joint pain is a common symptom in obese individuals and walking is often prescribed as part of management programs. Past studies in obese individuals have focused on standing alignment and kinematics in the sagittal and coronal planes. Investigation of 6 degree-of-freedom (6DOF) knee joint kinematics during standing and gait is important to thoroughly understand knee function in obese individuals with knee pain. This study aimed to investigate the 6DOF knee joint kinematics in standing and during gait in obese patients using a validated fluoroscopic imaging system. Ten individuals with obesity and knee pain were recruited. While standing, the knee was in 7.4±6.3°of hyperextension, 2.8±3.3° of abduction and 5.6±7.3° of external rotation. The femoral center was located 0.7±3.1mm anterior and 5.1±1.5mm medial to the tibial center. During treadmill gait, the sagittal plane motion, i.e., flexion/extension and anterior-posterior translation, showed a clear pattern. Specifically, obese individuals with knee pain maintained the knee in more flexion and more anterior tibial translation during most of the stance phase of the gait cycle and had a reduced total range of knee flexion when compared to a healthy non-obese group. In conclusion, obese individuals with knee pain used hyperextension knee posture while standing, but maintained the knee in more flexion during gait with reduced overall range of motion in the 6DOF analysis.