Spiders spin a great diversity of silk types for daily survival and reproduction. Of the six orb-weaver silk types, the dragline silk forming orb web frame attracts the most attention because of its extremely high tensile strength and toughness. So far, four types of major ampullate silk proteins (MaSp1-4) that make up dragline silk have been identified. These MaSp types have diversified amino acid motifs that underlie the impressive mechanical property of dragline silk by forming particular structures. Existing knowledge of MaSp4 proteins is fragmented, making it difficult to illuminate the structure and function of MaSp4. Here, we report the full-length MaSp4 gene with 11,334 bp from the orb-weaving spider Araneus ventricosus. Removing the only intron, the spliced complete transcript of MaSp4 gene is 6897 bp and encodes 2298 amino acids. Analysis of the primary structure of A. ventricosus MaSp4 protein reveals the repetitive region lacks poly-A and GGX motifs but has the unique GPGPQ motifs. Quantitative real-time PCR analyses show high levels of MaSp4 mRNA were detected in major ampullate gland. Structural characterization using CD- and FTIR sepctroscopy reveals a mainly α-helical solution conformation and a very high β-turn content within fibers. Collectively, our new findings provide complete template for recombinant silk protein with specific properties and support that the GPGPQ motif found in MaSp4 could increase flexibility in dragline silk by packing in more β-turns, expanding the repertoire of sequences known to form β-turn that is available for artificial chimeric silk fibers. STATEMENT OF SIGNIFICANCE: Dragline silk forming orb web frame attracts the most attention because of its extremely high tensile strength and toughness. So far, four types of major ampullate silk proteins (MaSp1-4) that make up dragline silk have been identified. Existing knowledge of MaSp4 proteins is fragmented, making it difficult to illuminate the structure and function of MaSp4. Here, we report the full-length MaSp4 gene from the orb-weaving spider Araneus ventricosus. We further identify the sequence, structure, and mechanical property of MaSp4 protein, providing a new insight into the structure-funtion relationships associated with MaSp4. Collectively, our new findings provide complete template for recombinant silk protein with specific properties and support that the GPGPQ motif found in MaSp4 could increase flexibility in dragline silk by packing in more β-turns, expanding the repertoire of sequences known to form β-turn that is available for artificial chimeric silk fibers.