Context: Ankle sprains are among the most common musculoskeletal injuries, and many individuals with ankle sprains develop chronic ankle instability (CAI). Individuals with CAI exhibit proprioceptive and postural-control deficits, as well as altered osteokinematics, during gait. Neuromuscular activity is theorized to play a pivotal role in CAI, but deficits during walking are unclear.Objective: To compare motor-recruitment patterns as demonstrated by surface electromyography amplitudes between participants with CAI and healthy control participants during walking.Design: Descriptive laboratory study. Setting: Laboratory.Patients or Other Participants: Fifteen adults with CAI (5 men, 10 women; age ¼ 23 6 4.2 years, height ¼ 173 6 10.8 cm, mass ¼ 72.4 6 14 kg) and 15 matched healthy control adults (5 men, 10 women; age ¼ 22.9 6 3.4 years, height ¼ 173 6 9.4 cm, mass ¼ 70.8 6 18 kg).Intervention(s): Participants walked shod on a treadmill while surface electromyography signals were recorded from the anterior tibialis, peroneus longus, lateral gastrocnemius, rectus femoris, biceps femoris, and gluteus medius muscles.Main Outcome Measure(s): Preinitial contact amplitude, postinitial contact amplitude, time of activation relative to initial contact, and percentage of activation time across the stride cycle were calculated for each muscle.Results: Time of activation for all muscles tested occurred earlier in the CAI group than in the control group. The peroneus longus was activated for a longer duration across the entire stride cycle in the CAI group (36.0% 6 10.3%) than the control group (23.3% 6 22.2%; P ¼ .05). No differences were noted between groups for measures of electromyographic amplitude at either preinitial or postinitial contact (P . .05).Conclusions: We identified differences between the CAI and control groups in the timing of muscle activation relative to heel strike in multiple lower extremity muscles and in the percentage of activation time across the entire stride cycle in the peroneus longus muscle. Individuals with CAI demonstrated neuromuscular-activation strategies throughout the lower extremity that were different from those of healthy control participants. Targeted therapeutic interventions for CAI may need to be focused on restoring normal neuromuscular function during gait.