The squid pen (gladius) from the Loligo vulgaris was used for preparation of β-chitin materials characterized by different chemical, micro-and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The β-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the β-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the β-chitin is modified, due to the intercalation of water molecules between β-chitin sheets. Potential applications of these β-chitin materials OPEN ACCESS Mar. Drugs 2014, 12 5980 range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications.