To examine the mechanism of the network formation of silk fibroin (SF), monodispersed colloidal particles (MDCPs) are used as well defined foreign substrates to quantify their effect on the primary nucleation of β-crystallites in molecular networks (silk nanofibrils) and the hierarchical network formation of SF. It follows that MDCPs are capable of accelerating the SF gelation by reducing the multistep nucleation barrier, which gives rise to a high density of silk fibril domain networks due to the increase of primary nucleation sites. Consequently, through governing the change in the hierarchical mesoscopic structure, the macroscopic performance of silk materials (e.g., the rheological properties of SF hydrogels and the tensile stress of fibers) can be controlled directly. As SF hydrogels represent a typical example of weak fibril domain–domain network interactions, the increase of fibril domain density leads to weaker gels. On the other hand, SF fibers correspond to strong fibril domain–domain network interactions, the increase of fibril domain density ends up with much tougher fibers. The knowledge obtained provides a facile strategy in controlling the complex hierarchical structure and macroscopic performance of SF materials, and offers useful routes for general design and functionalization of soft materials.