Antheraea yamamai silk contains long poly(alanine) sequences that contribute to β-sheet crystals, which are responsible for the high tensile strength of silk fibers. We determined the crystal structure, physical properties, and morphology of A. yamamai cocoon silk fibers. The crystal lattice of A. yamamai silk fiber consists of antiparallel β-pleated sheets with an orthogonal unit cell (a = 10.72 Å, b = 9.73 Å, c fiber axis = 6.80 ± 0.05 Å). Wide-angle X-ray scattering and birefringence measurements revealed that stretching deformation does not affect the crystal structure but contributes to alignment of silk molecules along the fiber axis in the partial amorphous phase. Crystallinity and amino acid sequence analyses suggested that the poly(alanine) region can be partially crystallized. The long poly(alanine) sequences therefore do not contribute to the mechanical, thermal, or structural properties of A. yamamai silk. Our results may aid the design and development of A. yamamai silk-based materials. Display omitted •The crystal structure of Antheraea yamamai silk were investigated.•The mechanical and thermal properties of A. yamamai silk were characterized.•The poly(alanine) sequence was partially crystalized in the beta-sheet crystals.