Heat shock protein 25 (Hsp25) phosphorylation plays a protective role following mechanical stress in skeletal muscle fibers. We previously reported that phosphorylation at serine 15 of Hsp25 (p-Ser15) was enhanced during regrowth of muscle fibers in rats with muscle atrophy due to tail suspension. However, it is still unclear how p-Ser15 contributes to myogenesis and regeneration of skeletal muscle fibers. We performed the present study to investigate p-Ser15 levels at different stages of myogenic differentiation in regenerating soleus muscle fibers of adult rats. Muscle regeneration was induced by muscle injury with intramuscular injection of cardiotoxin into the soleus. On day 14 after injury, p-Ser15-positive cells were noted in the regenerating soleus. The nuclei in small p-Ser15-positive cells contained myogenin, but not Pax7. Most of these cells did not exhibit peripheral localization of dystrophin, indicating that these cells were myotubes or immature fibers. Desmin and actinin were present in all cells and fibers regardless of p-Ser15 expression. Forced contraction by nerve stimulation led to increased phosphorylation at Ser15 in the regenerating soleus, as determined by western blot. Furthermore, elevated p-Ser15 was noted particularly in small cells with cross sectional areas less than 300 µm². These results suggest that small immature fibers are responsive to muscle contraction, and subsequently induce a protective response through the phosphorylation of Hsp25.