Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage. © Mary Ann Liebert, Inc.