Temperature-triggered phase separation of recombinant proteins has offered substantial opportunities in the design of nanoparticles for a variety of applications. Herein, the temperature-triggered phase separation behavior of a recombinant hydrophilic resilin-like polypeptide (RLP) is described. The transition temperature and sizes of RLP-based nanoparticles can be modulated based on variations in polypeptide concentration, salt identity, ionic strength, pH, and denaturing agents, as indicated via UV-Vis spectroscopy and dynamic light scattering (DLS). The irreversible particle formation is coupled with secondary conformational changes from a random coil conformation to a more ordered β-sheet structure. These RLP-based nanoparticles could find potential use as mechanically-responsive components in drug delivery, nanospring, nanotransducer, and biosensor applications.