20-Hydroxyecdysone (20E) is an ecdysteroid hormone that regulates moulting in insects. Interestingly, 20E is also found most abundantly in plant species and has anabolic effects in vertebrates, i.e. increasing muscle size without androgen influence. The effect of 20E on slow and fast fiber types of skeletal muscle has not been reported yet. Here we present that 20E affects the size (cross-sectional area, CSA) of the different fiber types in a muscle-specific manner. The effect on fiber size was modified by the distance from the site of the treatment and the presence of a regenerating soleus muscle in the animal. Besides the fiber size, 20E also increased the myonuclear number in the fibers of normal and regenerating muscles, suggesting the activation of satellite cells. According to our results 20E may provide an alternative for substitution of anabolic-androgenic steroids in therapeutic treatments against muscle atrophy.