Spider silk, known for its exceptional mechanical properties, is a sustainable protein-based material. However, recombinant spider silk proteins (spidroins) currently produced through biotechnological methods fall short in terms of mechanical performance compared to natural spider silk. This study reveals that spiders use a structural conversion of molecular enhancers—conserved globular 127-residue spacer domains—to create strong silk fibers. These domains, which lack poly-Ala motifs but contain motifs similar to human amyloidogenic motifs, self-assemble into amyloid-like fibrils through a non-nucleation-dependent pathway, likely to avoid cytotoxic intermediates. Incorporating this spacer domain into recombinant chimeric spidroins facilitates the self-assembly into silk-like fibers, increases molecular homogeneity, and significantly enhances fiber mechanical strength. The findings highlight that spiders employ diverse strategies to produce silk with exceptional mechanical properties, and the spacer domain offers a promising approach to enhance the properties of recombinant spider silk-like fibers and other functional materials.Spider silk, known for its exceptional mechanical properties, is a sustainable protein-based material. However, recombinant spider silk proteins (spidroins) currently produced through biotechnological methods fall short in terms of mechanical performance compared to natural spider silk. This study reveals that spiders use a structural conversion of molecular enhancers—conserved globular 127-residue spacer domains—to create strong silk fibers. These domains, which lack poly-Ala motifs but contain motifs similar to human amyloidogenic motifs, self-assemble into amyloid-like fibrils through a non-nucleation-dependent pathway, likely to avoid cytotoxic intermediates. Incorporating this spacer domain into recombinant chimeric spidroins facilitates the self-assembly into silk-like fibers, increases molecular homogeneity, and significantly enhances fiber mechanical strength. The findings highlight that spiders employ diverse strategies to produce silk with exceptional mechanical properties, and the spacer domain offers a promising approach to enhance the properties of recombinant spider silk-like fibers and other functional materials.