Molluscs are one of the most diversified phyla among metazoans. Most of them produce an external calcified shell, resulting from the secretory activity of a specialized epithelium of the calcifying mantle. This biomineralization process is controlled by a set of extracellular macromolecules, collectively defined as the organic matrix. In spite of several studies, these components are mainly known for bivalve and gastropod classes. In the present study, we investigated the physical and biochemical properties of the internal planispiral shell of the understudied Ram's Horn squid Spirula spirula (Cephalopoda, Decabrachia, Spirulida). Scanning Electron Microscope investigations of the shell reveal a complex microstructural organization, with septa sandwiched into the shell wall, in the form of a bevel. The saccharides constitute a quantitatively important moiety of the matrix, as shown by Fourier-transform infrared spectroscopy. Solid-state nuclear magnetic resonance spectroscopy identified β-chitin and additional polysaccharides, for a total amount of 80% of the insoluble fraction. Proteomics was applied to both soluble and insoluble matrices and in silico searches were performed, first on heterologous metazoans models, and secondly, on an unpublished transcriptome of Spirula spirula. In the first case, several peptides were identified, some of them matching with tyrosinase, chitinase 2, protease inhibitor, or immunoglobulin. In the second case, 38 hits were obtained, including transferrin, a serine protease inhibitor, matrilin, different histone-like, a 2-macroglobulin or a putative heme-binding/calcium-binding protein. The very few similarities with known molluscan shell matrix proteins suggest that Spirula spirula uses a unique set of shell matrix proteins for constructing its internal shell. The absence of similarity with closely related cephalopods such as the cuttlefish Sepia demonstrates that there is no obvious phylogenetic signal in the skeletal matrix of cephalopods.