Background: The purpose of this study was to investigate the differences in the muscle ac-tivity and co-activation index (CoA) of the rectus femoris (RF), biceps femoris (BF), gastrocnemius medialis (GM,) and tibialis anterior (TA) during walking on land and in water in healthy adolescents compared with those with spastic diplegia cerebral palsy (CP) adolescents. Methods: Four healthy individuals (median; age: 14 years, height: 1.57 cm, BMI: 16.58 kg/m2) and nine CP individuals (median; age: 15 years, height: 1.42 cm, BMI: 17.82 kg/m2) participated in this study and performed three walking trials under both conditions. An electromyography (EMG) collection was recorded with a wireless system Cometa miniwave infinity waterproof device, and the signals were collected using customized software named EMG and Motion Tools, Inc. software version 7 (Cometa slr, Milan, Italy) and was synchronized with an underwater VDO camera. Results: A significant decrease in the muscle activity of all muscles and CoA of RF/BF muscles, but an increase in TA/GM was observed within the CP group while walking in water during the stance phase. Between groups, there was a lower CoA of RF/BF and a greater CoA of TA/GM during the stance phase while walking in water and on land in the CP group. A non-significant difference was observed within the healthy group. Conclusion: Walking in water can decrease muscle activity in lower limbs and co-activation of thigh muscles in people with spastic CP, whereas CoA muscles around ankle joints increased to stabilize foot weight acceptance.