Background. Intrathecal baclofen (ITB) bolus injection effectively decreases spinal excitability but the impact on lower limb muscle activation during gait has not been thoroughly investigated. Objective. Examine activation of medial gastrocnemius (MG) and tibialis anterior (TA) muscles during gait before and after ITB bolus injection in patients with resting hypertonia after acquired brain injury. Methods. Lower extremity Ashworth score, temporospatial gait parameters, characteristics of the linear relationship between electromyogram (EMG) and lengthening velocity (LV) in MG during stance, and the duration and magnitude of TA-MG coactivation were assessed before and at 2, 4, and 6 hours after a 50-µg ITB injection via lumbar puncture in 8 hemorrhagic stroke and 11 traumatic brain injury subjects. Results. Temporospatial gait parameters did not significantly differ across the evaluation points (P ≥ .170). However, Ashworth score (P < .001), frequency and gain of significant positive EMG-LV slope (P ≤ .020), and duration of TA-MG coactivation (P ≤ .013) significantly decreased in the more-affected leg after ITB bolus. EMG changes were not significantly different between patients who did (n = 10) and did not (n = 9) increase gait speed after the injection. The timing of the largest decrease in Ashworth score and the largest decrease in EMG parameters coincided in 36% of cases, on average. Conclusions. ITB bolus injection alters the activation of MG and TA during gait. However, the changes in muscle activation are not closely related to the changes in gait speed or resting muscle hypertonia. The analysis of ankle muscle activation during gait better characterizes the response to ITB bolus injection than gait kinematics.