Despite the recognised role of the poly(lactide-co-glycolide) (PLGA) in the encapsulation and release of proteins from PLGA microspheres, the importance that the characteristics of the protein have in these processes has not yet been sufficiently investigated. The aim of this work was to study the simultaneous effect of the protein and PLGA properties and of the microencapsulation process on the physicochemical and in vitro release characteristics of protein-loaded PLGA microspheres. Two model proteins of different isoelectric points (pI), bovine serum albumin (BSA) (pI = 4.6) and lysozyme (LZM) (pI = 11.2), and two different molecular weights (Mw) of PLGA were selected. Microspheres were prepared using the water-in-oil-in-water (w/o/w) solvent extraction and the oil-in-oil (o/o) solvent evaporation techniques. Results showed that BSA was efficiently encapsulated independent of the PLGA Mw, whereas the encapsulation of LZM was favoured with the low Mw PLGA. The co-encapsulation of a surfactant (poloxamer 188 or 331) reduced the protein encapsulation efficiency, especially of BSA. These results suggested that the tensoactive properties of the protein and its affinity for the PLGA are major determinants of the protein encapsulation. Both proteins released faster from the microspheres prepared by the o/o solvent evaporation procedure, with respect to those prepared by the w/o/w solvent extraction technique. In addition, both polymer Mw and protein type had an effect on the protein release rate. The release rate of both proteins, in the absence of a surfactant, was faster from the low Mw PLGA microspheres. However, the release rate constant was higher for BSA than for LZM irrespective of the PLGA Mw. In addition, the co-encapsulation of a surfactant led, in most cases, to a faster release of the encapsulated protein. To conclude, these results suggest that protein release from PLGA microspheres is not only governed by the PLGA erosion rate and protein diffusion through the water-filled channels, but is highly affected by the protein properties and its possible interaction with PLGA and its degradation products.