[Purpose] This study determined the difference between flatfeet and normal feet in humans on an ascending slope using electromyography (EMG). [Subjects] This study was conducted on 30 adults having normal feet (n=15) and flatfeet (n=15), all of whom were 21 to 30 years old. [Methods] A treadmill (AC5000M, SCIFIT,) was used to analyze kinematic features during gait. These features were analyzed at slow, normal, and fast gait velocities on an ascending slope. A surface electromyogram (TeleMyo 2400T, Noraxon Co., USA) was used to measure muscle activity changes. [Results] The activities of most muscles in the subjects with flatfeet were significantly different from the muscle activities in the subjects with normal feet at different gait velocities on an ascending slope. There were significant differences in the vastus medialis and abductor hallucis muscles. [Conclusion] Because muscle activation of the vastus medialis in relation to stability of the lower extremity has a tendency to increase with an increase in gait velocity on an ascending slope, we hypothesized that higher impact transfer to the knee joints occurs in subjects with flatfeet due to the lack of a medial longitudinal arch and that the abductor halluces muscles, which provide dynamic stability to the medial longitudinal arches, do not activate well when they are needed in subjects with flatfeet.