In reorganization process of the actin stress fibers, a change in its mechanical condition has been suggested as one of the key mediators. Some experimental studies have clarified that tension release in the stress fibers induces the fiber depolymerization that is considered as the initial phase of its reorganization. However, quantitative mechanical values such as strain or stress that induce the depolymerization still have not been evaluated. This study tried to quantify the fiber strain that induces the depolymeridzaion of the actin stress fibers to gain a basic understanding of the reorganization phenomenon from a mechanical viewpoint. Osteoblastic cells were cultured on prestretched silicone rubber substrate. Compressive deformation was applied to the cells by uniaxially releasing the prestretched substrate strain and the change of the stress fiber structure was observed. The results indicated that the compressive strain, not in the whole cell body but in the stress fiber itself, is important to induce the disassembly of the fiber structure. In addition, the existence of a threshold strain for initiating fiber disassembly was suggested.