Fibronectin is a modular extracellular matrix protein involved in cell adhesion, cell motility, wound healing, and maintenance of cell morphology. It is composed of multiple repeats of three distinct modules: F I , F II , and F III . Various combinations of these modules create fragments able to interact with different constituents of the extracellular matrix. Here, we present the 2.5-Å resolution crystal structure of its 45-kDa gelatin-binding domain (GBD; 6F I -1F II -2F II -7F I -8F I -9F I ), which also corresponds to the C-terminal half of the migration stimulating factor, a Fn splice variant expressed in human breast cancers. GBD forms a very compact zinc-mediated homodimer, in stark contrast with previous structures of fibronectin fragments. Most remarkably, 8F I no longer adopts the canonical F I fold but is composed of two long strands that associate with 7F I and 9F I into a large β-sheet superdomain. Binding studies in solution confirmed that Zn induces conformational rearrangements and causes loss of binding of Fn-GBD to high-affinity collagen peptides. These data suggest the Zn may play a regulatory role for the cellular functions of fibronectin.