Objective To study the influence of different routes of keyhole limpet hemocyanin (KLH) immunization on the T-cell-dependent antibody response in mice.Methods SPF Kunming mice were divided into four groups:the intravenous injection group, subcutaneous injection group, intraperitoneal injection group and control group. Each mouse was injected 200μg KLH intravenously, subcutaneously or intraperitoneally daily for consecutive 10 days, respectively. Mice in the control group were given solvent injection only. Serum concentration of IgG stimulated by KLH antigen was measured 7 days after the last dosing. Spleen was isolated to calculate the organ coefficient and examined by pathology using hematoxylin and eosin staining.Results Intravenously, subcutaneously and intraperitoneally administered KLH stimulated the generation of secondary lymphoid follicles and germinal center to varying degrees, B cell apoptosis, increased amount of cells in the marginal zone and other pathological changes were observed in the spleen. Intravenous and intraperitoneal administration of KLH led to more pronounced pathological changes compared with that in the subcutaneous injection group. All of the three administration routes of KLH induced generation of IgG antibody, significantly higher than that in the control group (P<0.05). Intravenous injection of KLH generated the highest concentration of IgG and organ coefficient among the three administration routes (P<0.05). Conclusions Different immunization routes do affect the production of IgG antibody, organ coefficient and pathological changes in the spleen, and these differences should be taken into consideration when analyzing the T cell dependent antibody response in mice.