Aortic wall changes dimensions and mechanical properties in response to mechanical stimulation. As these changes are driven by the cells inside the wall, and their mechanical response has been suggested to exhibit a close correlation with nuclear deformation, it is necessary to study the deformation of the aortic wall at microscopic level. Hence, we obtained 200-µm-thick slices of rabbit thoracic aortas in the circumferential-radial and longitudinal-radial planes, and stretched them in the circumferential and longitudinal directions, respectively, under a microscope. The nuclei of smooth muscle cells (SMCs) were stained with Hoechst33342. Each slice was repeatedly stretched stepwise by 4%, while the fluorescence images of the cell nuclei as well as the elastin auto-fluorescence were captured at each step. Macroscopic and microscopic stretch ratios were obtained from the fluorescence images. Local Green strain was calculated from the change in internuclear distance in a specimen stretched in the circumferential direction. The local tissue strain in the circumferential direction was 0.8 to 2.1 times the macroscopic tissue strain, indicating that the aortic wall deformation was heterogeneous at microscopic level. The shear deformation between adjacent elastic laminas was evident at specific locations, resulting in a shear strain as large as 10%. We also evaluated the relationship between tissue deformation and nuclear deformation from the change in nuclear shape in the specimen stretched in the circumferential and longitudinal directions. In the circumferential stretch, the strain calculated from the length of the nuclei was less than 70% of the macroscopic strain, suggesting that the nuclei of the SMCs are much stiffer than the cytosolic components. Some nuclei rotated noticeably in response to the stretch, and the average and maximum rotation angle was 5° and 11°, respectively, during the entire stretching process. In the longitudinal stretch, the change in nuclear length was not significant, suggesting that mechanical stimulation to the SMCs may be smaller in this direction, as reported previously. The present study shows that the deformations of both the extracellular matrix and cell nuclei are highly heterogeneous, which may have a profound effect on the vascular biology.