The interaction between CD40 ligand (CD40L) and CD40 on antigen-presenting cells is essential for the initiation of antigen-specific T-cell responses. In order to clarify whether the expression of CD40L in tumor cells might be useful as a systemic therapy against bladder cancer, we investigated the antitumor immunity induced by CD40L in the mouse bladder cancer cell line MBT2. MBT2 was transduced by the retroviral vector expressing CD40L (MBT2-CD40L). Mouse bone marrow-derived dendritic cells cocultured with MBT2-CD40L cells produced eight times more IL-12 than those cocultured with parental MBT2 cells. In animal studies, subcutaneously inoculated MBT2-CD40L cells were rejected promptly. The vaccination of MBT2-CD40L cells induced antitumor immunity against parental tumors at a distant site. However, the antitumor effect of MBT2-CD40L inoculation was insufficient against pre-existing tumors. In the vaccination model, antibody ablation studies revealed that CD4(+) T cells were required for antitumor immunity, and tumor-specific cytotoxicity of sera was demonstrated. These data demonstrated that the antitumor immunity induced by CD40L was effective in the vaccination model and suggested that immunogene therapy using CD40L may be a new strategy of systemic therapy against bladder cancer.