Silk fibers featuring exceptional mechanical strength and high extensibility are composed of fibril bundles with diameters in the range of 20150 nm. Regeneration of silk fibers with similar superstrong mechanical properties from reconstituted silk protein remains a challenge because controlled self-assembly of macromolecular components on the nanoscale is required. The self-assembly of silk protein in nanoconfined geometry and the mechanical properties are investigated in this study. Using a template-filling method, cylindrical nanofibers of silk protein are fabricated for different diameters within nanopores (50120 nm) of anodic alumina oxide. By exposing to organic solvent, e.g. methanol, the proteins self-assemble to beta-sheet crystals in which the c-axis is aligned normal to the fiber axis, in stacking contrast to the natural silk fibers in which the c-axis is aligned along the fiber axis. Such highly ordered structures contribute to the enhanced mechanical property which reaches the theoretical mechanical level. In addition, the Youngs modulus of the nanofibers linearly increases with decreasing the diameter of the nanofibers. This is important, on the one hand, to understand the self-assembly of macromolecules under spatial confinement and, on the other hand, to understand the structureproperty relationships of nanomaterials and to fabricate soft nanostructures with controllable properties.