Uniaxially oriented, piezoelectric silk films are prepared by a two-step method that involves first air drying aqueous, regenerated silk fibroin solutions into films, and then drawing the silk films to a desired draw ratio. The utility of two different drawing techniques—zone drawing and water-immersion drawing—is investigated for processing the silk for piezoelectric studies. Silk films zone drawn to a ratio of λ 5 2.7 display relatively high dynamic shear piezoelectric coefficients of d14 5 –1.5 pC N21, corresponding to an increase in d14 of over two orders of magnitude due to film drawing. A strong correlation is observed between the increase in silk II, β-sheet content with increasing draw ratio as measured by FTIR spectroscopy (Cb e2.5λ), the concomitant increasing degree of orientation of β-sheet crystals detected via wide-angle X-ray diffraction (full width half maximum (FWHM) = 0.22° for λ = 2.7), and the improvement in silk piezoelectricity (d14 e2.4λ). Water-immersion drawing leads to a predominantly silk I structure with a low degree of orientation (FWHM 5 75°) and a much weaker piezoelectric response compared to zone drawing. Similarly, increasing the β-sheet crystallinity without inducing crystal alignment, e.g., by methanol treatment, does not result in a significant enhancement of silk piezoelectricity. Overall, a combination of a high degree of silk II, β-sheet crystallinity and crystalline orientation are prerequisites for a strong piezoelectric effect in silk. Further understanding of the structural origins of silk piezoelectricity provides important options for future biotechnological and biomedical applications of this protein.