Aquatic exercise is popular for training and rehabilitation, but information on how the water depth affects muscle activity when walking is lacking. The purpose of this study was to compare muscle activity when walking on land and at knee, pelvis and xiphoid process depth in a swimming pool. Twelve participants (22±3.6 years; 70.9±14.5kg; 1.7±0.1m) walked on land and on the pool floor at each of the three depths, at a self-selected (Vself) and a maximum speed (Vmax). Mean and peak muscle activity was recorded for the rectus femoris (RF), biceps femoris (BF), medial gastrocnemius (GM), lateral gastrocnemius (GL), tibialis anterior (TA) and erector spinae (ES). Stride duration was also recorded. Stride duration decreased and activity of all muscles increased from Vself to Vmax, except peak values at xiphoid process depth. For the depth comparisons, most changes in muscle activity occurred in the RF and BF, with higher values generally occurring at knee and pelvis depth, and stride duration continually increased with depth. These findings provide useful information on the musculoskeletal demands of walking in the water that can be used to inform design and prescription of exercise programmes for rehabilitation.