Changes in movement capabilities after an injury to the ankle may impose adaptations in the peripheral and central nervous system. The purpose of our study was to compare the electromyogram (EMG) profile of ankle stabilizer muscles and stride-time variation during treadmill running in individuals with and without chronic ankle instability (CAI). Recreationally active individuals with (n=12) and without (n=15) CAI ran on a treadmill at two speeds. EMG activity of four shank muscles as well as tibial acceleration data were recorded during the running trials. EMG amplitude, timing of EMG peaks, and variation in stride-time were analyzed from 30 consecutive stride cycles. EMG data were time-normalized to stride duration and amplitude was normalized relative to the appropriate maximal voluntary contraction (MVC) task. Individuals with CAI had similar EMG amplitudes and peak timing, but an altered order of peak EMG activity in ankle stabilizer muscles, a significantly greater EMG amplitude for PL with an increase in speed, and a greater stride-time variability during treadmill running compared with individuals who had no history of ankle sprains. The results of our study indicate that individuals with CAI exhibit altered activation strategies for ankle stabilizer muscles when running on a treadmill.