Context: Ankle instability is a common condition in physically active individuals. It often occurs during a jump landing or lateral motion, particularly when participants are fatigued. Objective: To compare muscle activation during a lateral hop prefatigue and postfatigue in individuals with or without chronic ankle instability (CAI). Design: Cross-sectional study. Setting: Sports medicine research laboratory. Patients or Other Participants: A total of 32 physically active participants volunteered for the study. Sixteen participants with CAI (8 men, 8 women; age = 20.50 +/- 2.00 years, height = 172.25 +/- 10.87 cm, mass = 69.13 +/- 13.31 kg) were matched with 16 control participants without CAI (8 men, 8 women; age = 22.00 +/- 3.30 years, height = 170.50 +/- 9.94 cm, mass = 69.63 +/- 14.82 kg) by age, height, mass, sex, and affected side. Intervention(s): Electromyography of the tibialis anterior, peroneus longus, gluteus medius, and gluteus maximus was measured before and after a functional fatigue protocol. Main Outcome Measure(s): Activation of 4 lower extremity muscles was measured 200 milliseconds before and after landing from a lateral hop. Results: We observed no interactions. The group main effects for the peroneus longus demonstrated higher muscle activation in the CAI group (52.89% +/- 11.36%) than in the control group (41.12% +/- 11.36%) just before landing the lateral hop (F-1,F-30 = 8.58, P = .01), with a strong effect size (d = 1.01). The gluteus maximus also demonstrated higher muscle activation in the CAI group (45.55% +/- 12.08%) than in the control group (36.81% +/- 12.08%) just before landing the lateral hop (F-1,F-30 = 4.19, P = .049), with a moderate effect size (d = 0.71). We observed a main effect for fatigue for the tibialis anterior, with postfatigue activation higher than prefatigue activation (F-1,F-30 = 7.45, P = .01). No differences were present between groups for the gluteus medius. Conclusions: Our results support the presence of a centralized feed-forward neuromuscular alteration in patients with CAI, not only in the ankle-joint muscles but also in the proximal hip muscles. These results may have implications for rehabilitation programs in these patients.