Conformation and activity control of proteins adsorbed on certain material surfaces enables the development of numerous high-performance applications. Herein, we examined the relationship between the diameter (surface shape) of polyurethane (PU) nanofibers and the conformation/activity of proteins adsorbed thereon, showing that hard segments align linearly in the long-axis direction when the PU structure is changed from random-segment to stretched nanofibers. Moreover, we revealed that the clustering of hydrophobic hard PU segments and protein adsorption are caused by hydrophobic interactions. Proteins adsorbed on thick nanofibers (diameter = 950 nm) showed decreased activity due to large conformational changes, whereas those adsorbed on thin nanofibers (diameter = 480 nm) retained a close-to-natural shape and thus showed relatively high activity, confirming that the shape of PU nanofiber surface affects the conformation and activity of proteins adsorbed thereon.