Spiders use structural conversion of globular amyloidogenic domains to create strong silk fibers. This study reveals that spiders utilize conserved globular 127-residue spacer domains, which lack poly-Ala motifs but contain motifs similar to human amyloidogenic motifs. These domains 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 enhances fiber molecular homogeneity and mechanical strength. The spacer domain is a natural amyloidogenic domain that spiders use to improve silk properties and direct fibrillization pathways. The findings highlight that spiders employ diverse strategies to produce silk with exceptional mechanical properties. The spacer domain offers a way to enhance the properties of recombinant spider silk-like fibers and other functional materials. Spidroins, which are synthesized and secreted by spider silk glands, undergo complex assembly and spinning processes to form silk fibers. Different spidroins have distinct sequences and properties, each used for specific silk or glue types. Recombinant spidroins, produced via recombinant DNA technology, can generate silk-like fibers but have lower mechanical strength compared to natural silks. The MiSp spacer domain, which is structurally related to type II spidroin repetitive regions, contains amyloid-forming motifs similar to those in human pathogenic amyloid peptides. The MiSp spacer domain can self-assemble into amyloid-like fibrils and facilitates the assembly of recombinant chimeric spidroins into silk fibers, significantly enhancing their mechanical strength. The study also shows that the MiSp spacer domain shares common motifs found in human pathogenic amyloidogenic peptides, and its presence in spider silk contributes to the formation of strong, homogeneous fibers. The findings suggest that the MiSp spacer domain is a natural amyloidogenic domain used by spiders to enhance silk properties while directing fibrillization pathways to prevent the formation of cytotoxic intermediates. The study concludes that the MiSp spacer domain is a natural biocompatible globular amyloidogenic domain that spiders use to enhance mechanical strength and holds great promise for the fortification of a wide range of functional materials.Spiders use structural conversion of globular amyloidogenic domains to create strong silk fibers. This study reveals that spiders utilize conserved globular 127-residue spacer domains, which lack poly-Ala motifs but contain motifs similar to human amyloidogenic motifs. These domains 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 enhances fiber molecular homogeneity and mechanical strength. The spacer domain is a natural amyloidogenic domain that spiders use to improve silk properties and direct fibrillization pathways. The findings highlight that spiders employ diverse strategies to produce silk with exceptional mechanical properties. The spacer domain offers a way to enhance the properties of recombinant spider silk-like fibers and other functional materials. Spidroins, which are synthesized and secreted by spider silk glands, undergo complex assembly and spinning processes to form silk fibers. Different spidroins have distinct sequences and properties, each used for specific silk or glue types. Recombinant spidroins, produced via recombinant DNA technology, can generate silk-like fibers but have lower mechanical strength compared to natural silks. The MiSp spacer domain, which is structurally related to type II spidroin repetitive regions, contains amyloid-forming motifs similar to those in human pathogenic amyloid peptides. The MiSp spacer domain can self-assemble into amyloid-like fibrils and facilitates the assembly of recombinant chimeric spidroins into silk fibers, significantly enhancing their mechanical strength. The study also shows that the MiSp spacer domain shares common motifs found in human pathogenic amyloidogenic peptides, and its presence in spider silk contributes to the formation of strong, homogeneous fibers. The findings suggest that the MiSp spacer domain is a natural amyloidogenic domain used by spiders to enhance silk properties while directing fibrillization pathways to prevent the formation of cytotoxic intermediates. The study concludes that the MiSp spacer domain is a natural biocompatible globular amyloidogenic domain that spiders use to enhance mechanical strength and holds great promise for the fortification of a wide range of functional materials.