Objective: To quantitatively analyze the in-vivo kinematics of the tibiofemoral joint in patients with arthrofibrosis after anterior cruciate ligament reconstruction (ACLR). Methods: Fifteen patients with arthrofibrosis after ACLR and 15 healthy subjects were included in the study. The tibiofemoral kinematics in 6 degrees of freedom during weightbearing and non-weightbearing conditions were collected using a portable knee 3D motion analysis system (Opti-knee?; Innomotion Inc, Shanghai, China). Results: Compared to healthy group, the internal tibial rotation was significantly decreased in patients with knee arthrofibrosis at 30°(p<0.001), 45°(p<0.001), 60°(p<0.001) and 75°(p<0.001) of weightbearing knee flexion; and tibial valgus was significantly decreased in the arthrofibrotic knee at 30°(p=0.002) and 45°(p=0.028) of weightbearing knee flexion. The internal tibial displacement in the arthrofibrotic knee was significantly reduced compared to healthy subjects at 75°(p=0.045) of non-weightbearing knee flexion. Conclusion: This study found that knee arthrofibrosis limits the rotation and internal displacement of the tibia. Therefore, in clinical practice, various treatments should be used to release the adhesions in the medial and lateral femoral grooves and the contracture of the lateral collateral ligament. It is critical to address the limitation of rotation and displacement movement in order to maximize the restoration of knee function.