AbstractA diurnal spider, Trichonephila clavata (T. clavata), is often called a golden orb‐weaver because its dragline or lifeline silk fibers exhibit a golden color. However, little is known about the biological roles of the golden pigment. Meanwhile, Araneus ventricosus, which is a nocturnal spider, spins colorless silk fibers. Here, we investigated the effect of ultraviolet (UV) irradiation on physical properties of silk fibers obtained from these diurnal and nocturnal spiders. After UV exposure, the dragline silk of the nocturnal spiders appeared abraded and became insoluble due to gelation, whereas the diurnal spider silk remained intact. A dynamic mechanical thermal analysis under different humidity conditions revealed that the storage modulus of the silk from the nocturnal spider dropped markedly at low humidity levels after UV irradiation. This indicated that the molecular weight of this silk degraded under UV exposure and that the number of terminal amino and carboxyl groups increased due to the breakage of the main protein chains. In contrast, the structural and mechanical properties of the diurnal spider silk were maintained regardless of the UV irradiation. Therefore, the golden pigments in the dragline silk of T. clavata most likely played a role in protecting the silk from UV damage.