Collagen is the most abundant protein in mammals and has found a wide range of applications in human health-related fields, namely cosmetics, pharmaceutical, dental, skin regeneration, ophthalmology, cardiac surgery and orthopaedics. Bovine and porcine bones and skins are the main industrial sources of collagen. However, due to religious constrains on the use of porcine products and to the risk of bovine spongiform encephalopathy (BSE) and other diseases posing to humans when infected bovine derived products are used, other sources of collagen are being pursued. Marine raw-materials have arisen on the last years and, in particular, marine sponges can be an important source of collagen. In this work, collagen was extracted from the marine sponge Chondrosia reniformis using different extraction methods: (i) 0.5 M acetic acid with 10% pepsin; (ii) 50 mM Tris-HCl with 1M NaCl ; and (iii) 100 mM Tris-HCl, 10 mM EDTA, 8 M urea and 100 mM 2-mercaptoethanol. Cytotoxicity of the extracted collagen was assessed with L929 cell culture onto collagen coatings (5.5µg/ml), with evaluation of metabolic activity (MTS assay) and cell proliferation (DNA quantification). Coatings of collagen extracted from C. reniformis are not cytotoxic and promoted proliferation. Moreover, C. reniformis collagen has been characterized and identified as mainly of type IV, thus promising application in epidermal regeneration strategies. In particular, its role on the selection of the rapid proliferating keratinocytes is addressed.