Proteins are nature's building blocks and indispensable in living organisms. Protein-based hydrogels have a wide variety of applications in research and biotechnology. In this chapter, we describe an intein-mediated protein hydrogel that utilizes two synthetic soluble protein block copolymers, each containing a subunit of a trimeric protein that serves as a cross-linker and one half of the naturally split DnaE intein from Nostoc punctiforme. Mixing of these two protein block copolymers initiates an intein trans-splicing reaction that constitutes a self-assembling polypeptide flanked by cross-linkers, triggering protein hydrogel formation. The generated hydrogels are highly stable under both acidic and basic conditions, and at temperatures up to 50 C. In addition, these hydrogels are able to undergo rapid reassembly after shear-induced rupture. Incorporation of an appropriate binding motif into the protein block copolymers enables the convenient site-specific incorporation of functional globular proteins into the hydrogel network.