The structure of native squid pen (gladius) was investigated in two different species on different length scales. By combining microscopy, atomic force microscopy (AFM), and X-ray diffraction, the experiments probed length scales from millimetres down to nanometres. The gladii showed a hierarchical, self-similar structure in the optical experiments with fibres of different size oriented along the long axis of the gladius. The fibre-like structure was reproduced at the nanoscale in AFM measurements and fibres with diameters of 500 μm, 100 μm, 10 μm, 2 μm and 0.2 μm were observed. Their molecular structure was determined using X-ray diffraction. In the squid gladius, the chitin molecules are known to form nano-crystallites of monoclinic lattice symmetry wrapped in a protein layer, resulting in β-chitin nano-fibrils. Signals corresponding to the α-coil protein phase and β-chitin crystallites were observed in the X-ray experiments and their orientation with respect to the fibre-axis was determined. The size of a nano-fibril was estimated from the X-ray experiments to be about 150 × 300 Å. About 100 of these nano-fibrils are needed to form a 0.2 μm thick micro-fibre. We found that the molecular structure is highly anisotropic with ∼90% of the α-coils and β-chitin crystallites oriented along the fibre-axis, indicating a strong correlation between the macroscale structure and molecular orientation.