Recombinant spider silk proteins mimicking the properties of dragline silk proteins represent a class of materials that hold great potential for future high-performance applications. Here we explore the self-assembly behavior of a recombinantly produced spider silk protein based on the dragline silk of the Araneus diadematus, eADF4 (C16), by selectively patterning its secondary structure using capillary transfer lithography and solvent-assisted microcontact molding. Two conformational transitions were observed, influenced by initial solvent composition: α-helix/random coil conformation to a more densely packed β-sheet conformation (by casting from 1,1,1,3,3,3-hexafluoro- propanol) and moderate initial β-sheet content to higher β-sheet content (casting from formic acid). Furthermore, by using the solvent-assisted microcontact molding technique, we were able to achieve a submicrometer spatial resolution and reveal fine details of morphological and mechanical changes in patterned regions and at interfaces.