Background: Multiple sclerosis (MS) is a progressive neurodegenerative disease characterized by axonal degeneration and demyelination. Changes in gait, related to joint kinematics and kinetics, especially at the ankle and knee, have been observed in people with MS (pwMS). Muscle coactivation plays an important role in joint stabilization; however, excessive coactivation may interfere with gait. The aim of this study was to analyze the differences in muscle activation during gait in pwMS compared to healthy individuals. Methods: A cross-sectional study was conducted involving pwMS and healthy controls. Surface electromyography was used to record muscle activity during gait. The main outcome measures were the coactivation index (CI) and the area under the curve (AUC), which were calculated for several pairs of lower extremity muscles. Results: Nine pwMS and nine healthy controls were included. When comparing the MS group to the control group, the AUC was significantly higher in the lateral gastrocnemius (p= 0.023) and the CI for the lateral gastrocnemius-anterior tibialis (p= 0.022) and gluteus maximus-lateral gastrocnemius (p= 0.047). Conclusion: Mildly affected pwMS have altered muscle coactivation patterns during gait, especially in the most affected limb. The results highlight the importance of muscle coactivation in pwMS and its possible role in the early detection of gait abnormalities.