Silk is a unique and exceptionally strong biological material. However, no synthetic method has yet come close to replicating the properties of natural silk. This shortfall is attributed to an insufficient understanding of both silk nanofibril structure and the mechanism of formation. Here in situ atomic force microscopy (AFM) and photo-induced force microscopy (PiFM) is utilized to investigate the formation process and define the basic structural paradigm of individual silk nanofibrils. By visualizing the multistage process of silk nanofibril formation, the importance of conformational transformations along the assembly pathway is revealed. Unfolded silk structures initially accumulate into amorphous clusters, which then evolve into crystal nuclei via conformational transformation into β-crystallites. Nanofibril elongation then occurs through the attachment of silk molecules at a single end of the nanofibril tip; this is facilitated through the formation of a new amorphous cluster that then repeats the aforementioned conformational transformation. However, enzymatic digestion of the amorphous regions leads to direct, rapid elongation of β-crystalline fibers. These findings imply that the energy landscape is characterized by shallow minima associated with intermediate states, which can be eliminated by introducing β-crystallites, and motivate research into the directed modification of the silk assembly pathway to select for features beneficial to specific applications.