Our study was the first to comprehensively examine the consequences of protein deprivation on variations in spider silk mechanics, structures, amino acid compositions and gene expressions at multiple scales across species. We concluded that: (i) MA silk properties of the spiders respond differently across multiple scales to variations in nutritional intake, and (ii) variations in spidroin expression and the crystalline and non-crystalline nanostructures play specific roles in inducing variations in the silk’s mechanical properties.