For the development of wearable and stretchable devices, insights into the mechanical properties and structural deformation of functional conjugated polymers are required. In particular, polythiophene has received much attention as a typical hole transfer material in electronic devices. However, the widely accepted polythiophenes are brittle because of the rigid chemical structure of thiophene rings. We have reported on the synthesis and flexible properties of polythiophene with disiloxane groups in side chains, and it was revealed that the polythiophene exhibited greater than 200% elongation at break at room temperature. In this study, we investigated the deformation process of polythiophene through in situ measurements under stretching using X-ray diffraction of synchrotron radiation and polarized infrared spectroscopy. In the X-ray diffraction measurements, orientation of the crystallites occurred after yielding, while the relative intensities of the polarized infrared absorption bands gradually increased during stretching. As seen from these results, during the initial deformation, the polythiophene chains in the amorphous region were aligned, and then, the whole bulk of the polythiophene, including crystallites and amorphous regions, were oriented after yielding. We succeeded in tracing the structural deformation of polythiophene during stretching. For the development of wearable and stretchable devices, stretchable and flexible semiconductive materials are desired. To understand the mechanical behavior of structural deformation of polythiophene with disiloxane groups, we performed in situ measurements under stretching using X-ray diffraction of synchrotron radiation and polarized infrared spectroscopy. The behaviors of amorphous region was oriented during the initial deformation, while the orientation of crystallites began after permanent set.