Although much is known about the mechanisms of anterior cruciate ligament (ACL) and other knee-related injuries, it is still unclear how chronic ankle instability (CAI) relates to these injury mechanisms. The purpose of the study, therefore, was to determine if individuals with CAI exhibit atypical knee biomechanics and muscle activation during landing onto a tilted surface. A seven-camera motion analysis system, two force plates and a surface electromyography (EMG) system were used to collect lower extremity biomechanics and EMG of 21 CAI and 21 pair-matched control (CON) participants who performed 10 landings onto a sideward-tilted and flat platform on the CAI/matched and non-test limbs, respectively. Kinematics (joint angles and displacement), kinetics (joint moments and eccentric work) and muscle activation (EMG linear envelope) were generated and compared between the CAI and CON groups using paired t-tests. CAI displayed an increased ankle inversion angle at initial contact; lower ankle inversion moment and eccentric work and increased EMG co-contraction during landing that could be related to their increased peak knee joint extension moment,internal rotation moment, and quadriceps-to-hamstring activation ratio during landing. This shows that CAI group successfully adapt their ankle landing mechanics to prevent the ankle from giving way by using greater ankle muscle co-contraction EMG to stabilize the foot. However, the increased co-contraction of ankle muscles could reduce the ankle energy dissipation in the frontal plane and further leaded to altered knee biomechanics and muscle activation. The atypical knee joint biomechanics and muscle activation (i.e., greater knee extension moment, internal rotation moment and quadriceps to hamstring co-contraction) of CAI group have been shown to be related to increased ACL loading. Future studies may need to measure/estimate the ACL loading to confirm that CAI relates to the mechanisms of ACL injury.