Siliceous sponges form their skeletal elements, the spicules, enzymatically via the sponge-specific enzyme silicatein. The enzymatic product of silicatein in vitro is a bio-silica polymer that is not processed/hardened by phase separation. In the present study we applied a two-phase system to investigate the transition of bio-silica, formed by silicatein, from the lucid state to the opaque/turbid state. We report for the first time that the polyether polyethylene glycol [PEG] causes a rapid transition of the lucid bio-silica to the opaque/turbid state. For the experiments the recombinant silicatein from the demosponge Suberites domuncula had been used. This process is rapid (1 h) and proceeds at ambient temperatures and low (<1 mM) ortho-silicate concentrations. The condensed material can be easily eye-inspected; it has been characterized by microscopy as a smooth and solid gel. The presence of PEG displays an accelerating effect on the enzymatic polycondensation reaction. In a second part of this study a natural, sponge protein has been isolated. The 22–24 kDa protein, termed the spicule-binding protein, causes a likewise increased sol–gel transition of bio-silica. The gene corresponding to this protein was identified in S. domuncula and found to encode for a protein containing the nidogen domain. The recombinant protein, the nidogen-related protein, was prepared and likewise found to induce gelation of bio-silica due to phase separation. It is proposed that the PEG-induced phase separation process follows a mechanism during which PEG together with silicatein neutralizes the negative surface charges of the formed bio-silica nanoparticles. Further on it is adopted that the phase separation process, caused by the nidogen-like protein (spicule-binding protein), can be ascribed best to a polymerization-induced phase separation process. It is concluded that the induction of the gelation process of bio-silica described here can be used during biomimetic fabrication of new bio-silica structures.