Acid-soluble (ASC) and pepsin-soluble (PSC) collagen were extracted from the skin of Nile tilapia (Oreochromis niloticus), purified and physicochemically examined. Amino acid content analyses revealed that glycine accounted for approximately one-third of the total amino acid residues. The proline and hydroxyproline contents of Nile tilapia ASC and PSC were 189 residues and 205 residues/1000 residues, respectively, and the rate of proline hydroxylation was found to be 41.8% and 42.0%, respectively. Denaturation temperatures (Td), as measured by an Ubbelohde viscometer, were 35.2°C and 34.5°C, respectively, 6°C lower than that of the type I collagen found in calf skin. In this study, we measured the intrinsic viscosity, circular dichroism (CD) and, X-ray diffraction (XRD), and employed Fourier transform infrared spectroscopy (FTIR) analyses to confirm that the ASC and PSC samples from Nile tilapia skin were native and undenatured, and therefore, maintained their original, intact triple helical structure. Our SDS-PAGE results showed that the extracted ASC and PSC peptides were in their native molecular form; (α)α (type I collagen). Furthermore, the loose, fibrous, and porous structures, shown in the cross-sections of ASC and PSC, indicate that Nile tilapia skin collagen represents a powerful physical foundation for further use in biomaterial applications.