Liposomal vesicles have been widely investigated as carriers for the intracellular delivery of oligonucleotides (ONs). To avoid unspecific uptake by the reticulo endothelial system, ‘pegylation’ of the liposomes, by incorporating polyethyleneglycol (PEG) at the surface, has been an attractive strategy. While pegylation has a clear benefit on the systemic level, one could wonder if pegylation also benefits the delivery efficacy of liposomes at the intracellular level. We compared the intracellular distribution of non-pegylated and pegylated liposomes, with special attention to the integrity of the oligonucleotides they are carrying. After uptake in the cells, the non-pegylated liposomes efficiently escaped from the endosomes thereby releasing phosphodiester oligonucleotides (PO-ONs) in the cytoplasm of the cells. The PO-ONs were however rapidly degraded in the intracellular environment. In contrast to non-pegylated liposomes, pegylated liposomes failed in protecting the PO-ONs they were carrying, leading to rapid degradation of the PO-ONs in the endosomal compartment. Furthermore, the PEG chains inhibited the endosomal escape of the degraded ONs. These intracellular findings explain why pegylated liposomes failed in establishing an antisense effect.