A silk-fiber matrix was studied as a suitable material for tissue engineering anterior cruciate ligaments (ACL). The matrix was successfully designed to match the complex and demanding mechanical requirements of a native human ACL, including adequate fatigue performance. This protein matrix supported the attachment, expansion and differentiation of adult human progenitor bone marrow stromal cells based on scanning electron microscopy, DNA quantitation and the expression of collagen types I and III and tenascin-C markers. The results support the conclusion that properly prepared silkworm fiber matrices, aside from providing unique benefits in terms of mechanical properties as well as biocompatibility and slow degradability, can provide suitable biomaterial matrices for the support of adult stem cell differentiation toward ligament lineages. These results point toward this matrix as a new option for ACL repair to overcome current limitations with synthetic and other degradable materials.