We have reported that PEGylated liposomes lose their long-circulating properties when injected twice into the same animal within a certain interval (the accelerated blood clearance (ABC) phenomenon). We assumed that this phenomenon was triggered via the abundant secretion of anti-polyethylene glycol (PEG) immunoglobulin M (IgM) in response to the first dose of PEGylated liposomes and that the spleen played an important role in the production of anti-PEG IgM. However, no direct evidence has yet confirmed this suspicion. In the current study, we verified, both in vitro and ex vivo , that spleen cells are indeed responsible for the production of anti-PEG IgM in response to PEGylated liposomes. In this study, spleen cells obtained from either naïve mice or mice pre-treated with PEGylated liposomes induced the production of anti-PEG IgM in a dose- and time-dependent manner, upon incubation with PEGylated liposomes. In addition, we confirmed that among the different fractions of splenic B cells, IgM-positive B cells, rather than CD45R-positive or CD19-positive splenic B cells, which are presumed to be the marginal zone B (MZB) cells, are the major cells producing anti-PEG IgM in the response to stimulation by PEGylated liposomes. These results may provide new insights into the mechanisms underlying the anti-PEG IgM production in response to the stimulation by PEGylated liposomes.