The local and global structural changes of cytochrome c induced by urea in aqueous solution have been studied using X-ray absorption spectroscopy (XAS) and small-angle X-ray scattering (SAXS). According to the XAS result, both the native (folded) protein and the unfolded protein exhibit the same preedge features taken at Fe K-edge, indicating that the Fe(III) in the heme group of the protein maintains a six-coordinated local structure in both the folded and unfolded states. Furthermore, the discernible differences in the X-ray absorption near-edge structure (XANES) of these two states are attributed to a possible spin transition of the Fe(III) from a low-spin state to a high-spin state during the unfolding process. The perseverance of six-coordination and the spin transition of the iron are reconciled by a proposed ligand exchange, with urea and water molecules replacing the methionine-80 and histidine-18 axial ligands, respectively. The SAXS result reveals a significant morphology change of cytochrome c from a globular shape of a radius of gyration R-g = 12.8 angstrom of the native protein to an elongated ellipsoid shape of R-g = 29.7 angstrom for the unfolded protein in the presence of concentrated urea. The extended X-ray absorption fine structure (EXAFS) data unveil the coordination geometries of Fe(III) in both the folded and unfolded state of cytochrome c. An initial spin transition of Fe(III) followed by an axial ligand exchange, accompanied by the change in the global envelope, is proposed for what happened in the protein unfolding process of cytochrome c.