This study characterizes a new cold-formed recombinant humanized collagen type III gel (C3Gel) using a multi-technique approach to analyze composition, structure, thermal properties and morphology. The molecular weight and structural sequence of C3Gel correspond to theoretical expectations. At low temperatures, C3Gel self-crosslinked into a porous network structure. The gel shows favorable cytocompatibility with HaCaT cells. It improved the extracellular matrix (ECM) and provided structural support to HaCaT cells. It facilitated the adhesion, proliferation and migration of these cells. C3Gel is promising as a new biomedical material for further research and applications.