Microspheres allowing the controlled release of the model oligonucleotide pdT16 were designed. The oligonucleotide, alone or associated with polyethylenimine (PEI) at different nitrogen/phosphate ratios, was encapsulated within poly(lactide-co-glycolide) microspheres prepared by the multiple emulsion-solvent evaporation technique. The introduction of PEI in the internal aqueous phase resulted in a strong increase of the oligonucleotide encapsulation efficiency. PEI affected also microsphere morphology inducing the formation of very porous particles and yielding to an accelerated release of pdT16. However, when incubated with HeLa cells, microspheres encapsulating pdT16/PEI complexes allowed an improvement of the intracellular penetration of the released oligonucleotide. The developed strategy appears to be a very interesting tool to obtain a sustained release system for oligonucleotides with an efficient cellular delivery.