Silk fibers featuring exceptionalmechanical strength and high extensibility are composed of fibrilbundles with diameters in the range of 20–150 nm. Regenerationof silk fibers with similar superstrong mechanical properties fromreconstituted silk protein remains a challenge because controlledself-assembly of macromolecular components on the nanoscale is required.The self-assembly of silk protein in nanoconfined geometry and themechanical properties are investigated in this study. Using a template-fillingmethod, cylindrical nanofibers of silk protein are fabricated fordifferent diameters within nanopores (50–120 nm) of anodicalumina oxide. By exposing to organic solvent, e.g. methanol, theproteins self-assemble to β-sheet crystals in which the c-axis is aligned normal to the fiber axis, in stackingcontrast to the natural silk fibers in which the c-axis is aligned along the fiber axis. Such highly ordered structurescontribute to the enhanced mechanical property which reaches the theoreticalmechanical level. In addition, the Young’s modulus of the nanofiberslinearly increases with decreasing the diameter of the nanofibers.This is important, on the one hand, to understand the self-assemblyof macromolecules under spatial confinement and, on the other hand,to understand the structure–property relationships of nanomaterialsand to fabricate soft nanostructures with controllable properties. [ABSTRACT FROM AUTHOR]