Materials that effectively deliver protein antigens together with activating ligands to antigen-presenting cells are sought for improved nonviral vaccines. To this end, we synthesized protein-loaded poly(ethylene glycol) (PEG)-based hydrogel particles by cross-linking PEG within the polymer-rich phase of an emulsion formed by a poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) triblock copolymer in saturated aqueous salt solution. These particles (500-nm diameter) contained high levels of encapsulated protein (similar to 75% of dry mass), which was selectively released by proteolytic enzymes normally present in the phagosomal/endosomal compartments of dendritic cells (DCs). For co-delivery of cellular activation signals, gel particles were surface-modified by sequential adsorption of poly(L-arginine) and CpG oligonucleotides. DCs pulsed with protein-loaded particles activated naive T cells in vitro similar to 10-fold more efficiently than DCs incubated with soluble protein. This organic solvent-free strategy for protein encapsulation within submicron-sized hydrophilic particles is attractive for macromolecule delivery to a variety of phagocytic and nonphagocytic cells.