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Metallic nanowires usually exhibit ultrahigh strength but low tensile ductility owing to their limited strain hardening capability. Here we study the unique strain hardening behavior of the five-fold twinned Ag nanowires by nanomechanical testing and atomistic modeling. In situ tensile tests within a scanning electron microscope revealed strong strain hardening behavior of the five-fold twinned Ag nanowires. Molecular dynamics simulations showed that such strain hardening was critically controlled by twin boundaries and pre-existing defects. Strain hardening was size dependent; thinner nanowires achieved more hardening and higher ductility. The size-dependent strain hardening was found to be caused by the obstruction of surface-nucleated dislocations by twin boundaries. Our work provides mechanistic insights into enhancing the tensile ductility of metallic nanostructures by engineering the internal interfaces …