Immobilization is often associated with a decrease in muscle elasticity. This condition is called muscle contracture, but the mechanism is not yet clear. We examined changes in ankle joint mobility, sarcomere length, collagen fibril arrangement in the endomysium, and hyaluronic acid (HYA) in muscular tissue 1, 2, 4, 8, and 12 weeks after immobilization of rat soleus muscles in shortened position. Ankle joint mobility decreased with the duration of immobilization. Sarcomere length had shortened 1 week after immobilization, but did not show further change 2, 4, 8, and 12 weeks after immobilization. Collagen fibril arrangement in the endomysium 1 and 2 weeks after immobilization was longitudinal to the axis of the muscle fibers, whereas 4, 8, and 12 weeks after immobilization it was circumferential. HYA in muscular tissue had increased 1 week after immobilization but remained at the same level at weeks 2, 4, 8, and 12. Histochemically, HYA in the endomysium of immobilized muscular tissue was stained more strongly and widely than that in the control tissue. Increased HYA in muscular tissue may induce muscle stiffness, but the significance of how HYA is related to the mechanism of muscle contracture was not clear. The findings suggest that muscle contracture started 1 week after immobilization and increased with the length of immobilization. Consequently, muscle contracture is affected by the shortening muscle fibers during the early stage of immobilization, after which the collagen adapts by the fibril arrangement in the endomysium becoming more circumferential. This change in collagen fibril arrangement may cause advanced muscle contracture in the late stage of immobilization.