Abstract: Objective To better evaluate the ergonomics of an ankle-foot orthosis (AFO), this paper proposes a method to quantify the impact of stiffness on gait by using the man-machine coupling model of AFO based on musculoskeletal simulation technology. Methods Firstly, the stiffness of two AFOs was quantified, and the kinetics and kinematics parameters of 30 subjects were collected when walking under normal and worn two different stiffnesses of AFOs. Secondly, a man-machine coupled model with AFO was created based on the OpenSim platform. Finally, the changes in the joint angle, joint torque, and main muscle force of the lower limbs were quantitatively analyzed by simulation under two different AFO stiffness. Results Under two different AFO stiffnesses, the peak dorsiflexion angle of the ankle, peak knee flexion angle, hip extend angle significantly increased (P＜0.05), and soleus and gastrocnemius peak muscle force also showed an increasing trend. The peak plantarflexion angle of ankle, hip flexion angle, and peak moment of hip flexion obviously decreased (P＜0.05). Conclusions The experimental results in which the effect of different AFO stiffness on gait was found by this paper similar to previous research findings. Thus, the AFO man-machine coupled simulation method was proposed in the research, which can provide a novel method for effectively quantitative evaluating the influence of AFO stiffness on gait biomechanical. Meanwhile, it can also be applied to assess and optimize the ergonomics of other assistive devices. In addition, the study also has a certain clinical significance and provides theoretical guidance for the selection, adaptation, and optimization design of AFO.