In this paper, the release of proteins from a novel self-gelling hydrogel based on biodegradable dextran microspheres is investigated. The protein-loaded macroscopic gels are obtained by hydration of mixtures of oppositely charged hydroxyethyl methacrylate-derivatized dextran microspheres with a protein solution. In media of low ionic strength (100 mM Hepes pH 7.0) it was found that the release of the entrapped model proteins (lysozyme, BSA and IgG) was slower than in saline (150 mM NaCl, 100 mM Hepes pH 7.0). The reason behind this observation is that substantial adsorption of the proteins onto the microspheres' surface and/or absorption in the microspheres takes place. Confocal images showed that independent of their crosslink density the microspheres are impermeable for BSA and IgG. BSA, bearing a negative charge at neutral pH, was adsorbed onto the surface of positively charged microspheres. Lysozyme, which is positively charged at neutral pH, was able to penetrate into the negatively charged microspheres. In saline, the gels showed continuous release of the different proteins for 25 to 60 days. Importantly, lysozyme was quantitatively and with full preservation of its enzymatic activity released in about 25 days. This emphasizes the protein friendly technology to prepare the protein-loaded gels. Mathematical modeling revealed that protein release followed Fick's second law, indicating that the systems are primarily diffusion controlled. These results show that these hydrogels are very suitable as injectable matrix for diffusion-controlled delivery of pharmaceutically active proteins.