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The present study aimed to investigate differences in muscle activation of peroneus longus (PL) and tibialis anterior (TA) among individuals with chronic ankle instability (CAI), copers, and heatlhy controls during walking and running. A total of 54 participants were recruited for this study. 54 participants were equally assigned to the CAI, coper, and control groups based on eligibility criteria for each group. Two surface electromyography (sEMG) were attached to PL and TA of the affected leg and 33 reflective markers were also attached to the landmark of participants based on the Visual 3D marker set. All participants were instructed to walk and run at the speed of 1.34 m/s with a boundary of 0.07 m/s and 2.68 m.s with a boundary of 0.14 m/s along the 8 m walkway. Three valid gait trials were recorded and analyzed. The full gait cycle from initial contact (IC) of the affected leg to next IC of the same leg were normalized to 100%. All sEMG data were normalized by the amplitude during maximum voluntary contraction of each participant. Ensemble curve analyses with mean ± 90% confidence intervals were conducted to identify mean differences in the amplitude of two muscles among three groups. The CAI group showed the greater amplitude of PL during walking than the coper and control groups during walking. Individuals with CAI exhibited the altered muscle activation of TA compared with copers and controls during walking. In addition, our findings indicated that there were significantly different in muscle activation of both of PL and TA between the CAI and non-CAI groups during running. Given our results, individuals with CAI need to improve joint stability using pre-activation before IC during gait.