Microbially prepared polypentapeptides were cross-linked by two chemical methods. In one chemical approach, (GVGIP)260 where G = glycine, V = valine, I = isoleucine, and P = proline with no functional groups in its side chains, was cross-linked using dicumyl peroxide, and reaction conditions were systematically examined. Successful cross-linking was obtained even under severe conditions for proteins, i.e., 3 h at 120 °C, without having significant side reactions. In the second chemical approach, two separate polymers, (GVGVP GVGVP GXGVP GVGVP GVGVP GVGVP)n where X is either E (the carboxylic acid containing glutamic acid residue) or K (the lysine residue with an ε-amino function), were mixed and cross-linked using a carbodiimde reagent. The reaction temperature was found to affect the equilibrium swelling behavior of the resulting cross-linked hydrogels. In hydrogels cross-linked at a temperature above their hydrophobic folding and assembling transition temperature, 3D continuous filamentous microstructures were observed. Chemically cross-linked hydrogel fibers were also prepared and their anisotropy in swelling was confirmed. Uniaxial tensile moduli and equilibrium weight swelling ratios of the chemically cross-linked samples were compared to those of (GVGVP)251 and (GVGIP)260, γ-irradiation cross-linked at different Mrad doses.