Splenic marginal zone B (MZ-B) cells have attracted attention as alternative antigen-presenting cells. We recently developed an original delivery system, using PEGylated liposomes (PEG-Lip) to deliver antigens to MZ-B cells. In this system, to induce antigen-specific immunity, empty PEG-Lip and antigen-containing PEG-Lip were intravenously (i.v.) injected sequentially at 3 day intervals. Since complement activation by the second dose is required for the delivery of antigen-containing PEG-Lip to splenic MZ-B cells, we investigated the ability of liposomes, modified with various PEG derivatives having different functional terminal groups (methoxy PEG (CH3O-PEG), hydroxy PEG (HO-PEG) or polyglycerol (PG), to activate the complement system and deliver a model antigen, ovalbumin (OVA), to splenic MZ-B cells in vitro and in vivo. Hydroxy PEG-modified liposomes (HO-PEG-Lip) both activated the complement system in vitro, and facilitated the preferential association of HO- PEG-lip with MZ–B cells in vitro. Manipulating HO-PEG density, in particular a density of 2 mol% in total lipids, significantly enhanced the association of HO-PEG-Lip with splenic MZ-B cells in vivo. Consequently, a single i.v. injection of HO-PEG-Lip (2 mol%) containing OVA induced OVA-specific IgG response. Our immunization system with HO-PEG-Lip, achieved efficient antigen delivery to MZ-B cells after a single i.v. injection, improving on our previous immunization system. This new delivery technique may be an improved, simple, antigen delivery system to MZ-B cells that induces meaningful levels of humoral immune response.