Changes in structure and properties due to mechanical fatigue for segmented polyurethanes containing a block segment of poly(ethylene oxide)-block-poly(dimethyl siloxane)-block-poly(ethylene oxide) (PES) were investigated by mechanical and thermal analyses and a small-angle X-ray scattering (SAXS) technique. Six types of specimens designated KP-x-y were studied, where x and y denote, respectively, the fraction of PES component (x = 0 or 13 wt%) and the molecular weight of the soft-segment block, poly(tetramethylene glycol) (PTMG) (y = 1000, 2000 or 3000). The SAXS experiments revealed that dissociation of the hard-segment microdomains was induced by mechanical fatigue. The changes in the mechanical properties due to fatigue were discussed in relation to the PTMG soft-segment crystallization which can be triggered by the dissociation of the hard-segment microdomains. As already reported, KP-13-2000 has better antithrombogenicity and more suitable viscoelastic properties for use as a biomedical material than any of the others. It was found in this study that KP-13-2000 has strong resistance against mechanical fatigue as well, which is one of the most important characteristics required for biomedical materials. Although the microdomain structure was fractured by fatigue, the mechanical property of KP-13-2000 was not changed because the PES component prevents the PTMG soft segments from crystallizing.