Steric stabilization of the surface of liposomes by a PEG conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended their circulation times (t1/2≈20h in rat). Recently, we reported on the “accelerated blood clearance phenomenon”, causing ‘invisible’ PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. In addition, we reported that certain serum factor(s) secreted into the blood after the first dose of PEGylated liposomes play an essential role in the phenomenon. The aim of the present study was to identify the major serum protein(s) responsible for the phenomenon and to unravel their mode of action. The amount of protein binding to PEGylated liposomes during incubation with serum hardly correlated with the extent of the phenomenon. PEGylated liposomes incubated with serum obtained from rats pre-injected 5 days before with the same liposomes showed a much more complex pattern of bound proteins than when incubated with naïve serum, as revealed by 2D-PAGE and SDS-PAGE. Subsequent analysis with LC-MS/MS and Western blot showed that the major pre-treated serum protein binding to PEGylated liposomes was IgM. Semi-quantitative analysis showed that larger amount of IgM bound to PEGylated liposomes compared to conventional liposomes. It was further demonstrated that PEGylated liposomes could activate the complement system due to IgM binding when incubated in serum from rats pre-injected with PEGylated liposomes, while conventional liposomes were not. These findings suggest that the selective binding of IgM to the second injected PEGylated liposomes and the subsequent complement activation by IgM resulted in the accelerated clearance and enhanced hepatic uptake of the second injected PEGylated liposomes. Based on the results described here, we are drawing attention to the potential occurrence of unexpected immune reactions upon intravenous administration of PEGylated liposomes or other particles and, by extension, PEGylated proteins and DNAs.