Purpose: The aim of the present study was to make an ankle destabilization device and investigate the effect of exercise with this device on peroneal muscles.Methods: Seventeen healthy subjects were instructed to undergo step movement exercise with the lower extremity 150 times on the spot using the ankle destabilization device. Before and after stepping, the reaction time and mean frequency of the peroneus longus and the peroneus brevis muscles were compared. While stepping with the device, muscular activity of the peroneus longus and the peroneus brevis was compared under both stable and instable conditions.Result: The results showed that peroneal reaction time didn't change after stepping with the device; but the frequency of peroneal muscle activity increased after stepping. While stepping, muscular activity of the peroneal muscles increased in an instable condition.Conclusion: The results suggested that stepping exercise with the device could be beneficial for peroneal muscles.