Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for the potential application in cartilage regeneration. The influence of collagen concentration, salinity, and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. Formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSC) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II, and aggrecan were upregulated in direct cultures of human mesenchymal stem cells (hMSC) upon chondrogenic stimulation. Formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycanes.