This study sought to determine the characteristics of gait patterns and muscle activity in the lower extremities of elderly women during underwater treadmill walking against water flow. Eight female subjects (61.4±3.9 y) performed underwater and land treadmill walking at varying exercise intensities and velocities. During underwater walking (water level at the xiphoid process) using the Flowmill, which has a treadmill at the base of a water flume, the simultaneous belt and water flow velocities were set to 20, 30 and 4 m·min⁻¹. Land walking velocities were set to 40, 60 and 80 m·min⁻¹. Oxygen uptake and heart rate were measured during both walking exercises. Maximum and minimum knee joint angles, and mean angular velocities of knee extension and knee flexion in the swing phase were calculated using two-dimensional motion analysis. Electromyograms were recorded using bipolar surface electrodes for five muscles: the tibialis anterior (TA), medial gastrocnemius (MG), vastus medialis (VM), rectus femoris (RF) and biceps femoris (BF). At the same exercise intensity level, cadence was almost half that on land. Step length did not differ significantly because velocity was halved. Compared to land walking, the maximum and minimum knee joint angles were significantly smaller and the mean angular velocity of knee extension was significantly lower. Knee extension in the swing phase was limited by water resistance. While the muscle activity levels of TA, VM and BF were almost the same as during land walking, those of MG and RF were lower. At the same velocity, exercise intensity was significantly higher than during land walking, cadence was significantly lower, and step length significantly larger. The knee joint showed significantly smaller maximum and minimum angles, and the mean angular velocity of knee flexion was significantly larger. The muscle activity levels of TA, VM, and BF increased significantly in comparison with land walking, although those of MG and RF did not significantly differ. Given our findings, it appears that buoyancy, lower cadence, and a moving floor influenced the muscle activity level of MG and RF at the same exercise intensity level and at the same velocity. These results show promise of becoming the basic data of choice for underwater walking exercise prescription.