Objective To investigate the mechanical responses, structural alterations, and constitutive characterization of femoral and popliteal arteries under biaxial stretching. Methods Femoral artery (FA) and popliteal artery (PA) samples were harvested from the lower limbs of 36-month-old male Wuzhishan minipigs weighing 36 kg. Planar biaxial tensile tests were conducted to obtain stress-strain data. Histological and ultrastructural changes in the vessel wall before and after stretching were evaluated using EVG staining, Masson staining, α-SMA immunohistochemistry, and transmission electron microscopy (TEM). Based on the biaxial experimental data, an improved four-fiber polynomial strain energy function model was employed to fit the constitutive parameters of FA and PA. Results Both FA and PA exhibited pronounced nonlinear stress-strain behavior under biaxial loading, with circumferential stress exceeding axial stress under equibiaxial loading. Before stretching, FA showed the highest α-SMA-positive area fraction (85.55%). After stretching, the elastic fiber area fraction decreased from 42.61% to 33.67% in FA and from 43.07% to 36.94% in PA, whereas the collagen fiber area fraction increased from 39.94% to 40.72% in FA and from 25.96% to 31.63% in PA. In both groups, smooth muscle-related structures underwent morphological alterations, and elastic fiber-related structures exhibited reduced continuity with a tendency toward localized discontinuity. TEM further revealed that ultrastructural differences between FA and PA were already present before stretching. Constitutive fitting demonstrated that the proposed model provided a better overall description for FA than for PA, with overall R^2 values of 0.8744 and 0.8716 in the axial and circumferential directions of FA, respectively, whereas the fitting accuracy in the circumferential direction of PA was relatively lower. Conclusion Both FA and PA exhibited nonlinear mechanical behavior under biaxial stretching, accompanied by alterations in vascular wall cells and extracellular matrix-related structures. Initial differences in tissue architecture, together with load-induced structural remodeling, may jointly contribute to their subsequent mechanical responses. In addition, constitutive characterization of the circumferential mechanical behavior of PA appears to be more challenging.