Films of regenerated silk fibroin (RSF) are usually brittle and weak, which prevents its wide application as a structural material. To improve the mechanical properties of RSF film, uniaxial extension under swollen conditions was employed to introduce preferred orientation of molecular chains of silk fibroin. Such a prestretching treatment resulted in the strain at break, ultimate stress, Young's modulus, and energy to break along the predrawn direction of the RSF film increasing from approximate 5%, 90 MPa, 2.7 GPa, and 2.1 kJ/kg to 35%, 169 MPa, 3.5 GPa, and 38.9 kJ/kg, respectively, which is an attractive combination of strength and toughness. The mechanism of these property enhancements was investigated using techniques such as small-angle X-ray scattering, wide-angle X-ray diffraction, atomic force microscopy, and dynamic mechanical analysis.