Background: Flat feet can be identified by assessing the collapse of the medial longitudinal arch (MLA) and these conditions can trigger epidemiological changes in the feet. Many of previous studies compared the muscle activity of lower body in terms of intervention and dynamics to treat the structural defect of flat feet. However, few studies have investigated or analyzed the muscle activity of gastrocnemius muscle in the subjects with flat feet. Objects: This study investigated the differences in changes of medial and lateral plantar flexors in subjects with flat feet during bipedal heel-rise (BHR) task and analyzed the differences in muscle activity between two groups by measuring the electromyography (EMG) of abductor hallucis (AH), tibialis anterior (TA), medial gastrocnemius (MG), lateral gastrocnemius (LG). Methods: A total of Twenty one adult females participated in this experiment. Subjects were assigned to groups according to the navicular drop test. The task was applied to the leg, where the heel lifting action prevailed. The muscle activity of the medial and lateral feet plantar flexors was evaluated, and the % maximum voluntary isometric contractions (%MVIC) of these were compared. Results: For the difference between groups the muscle activity (%MVIC) of LG muscle was statistically significantly low in flat feet group compared to healthy feet group (flat feet: 64.57, healthy feet: 90.17; p < 0.05). Conclusion: The results of this study will contribute to identifying the muscle activities of medial and lateral feet plantar flexors among subjects with flat feet, which can cause abnormal epidemiological changes in the feet.