Experimental autoimmune myocarditis (EAM) has been used as a model for human myocarditis in relation to the autoimmune mechanism and proved as a T cell-mediated autoimmune disease. Interaction of T cell receptors (TCR) with its ligand peptide-MHC complex on APCs is critical for antigen-specific T cell activation under physiological and pathological conditions. CIITA is a transcriptional coactivator that functions as a key regulatory factor for MHC-II expression. To achieve effective inhibition of interaction of TCR and peptide-MHC-II complex, adenovirus vectors containing antisense CIITA were constructed and their effects in preventing EAM were examined. Ad-CIITA was injected intravenously into mice on days 0-2 or 14-16 after myosin immunization to study the preventive effects on EAM in the T cell activation phase or inflammatory phase. Disease severity was determined by the microscopic grade of heart check, concentration of plasma cTnI, and cellular and humoral immune responses on day 21. Results show that onset of EAM after Ad-CIITA treatment on days 0-2 was almost completely inhibited and antigen-specific lymphocyte proliferation was significantly reduced in adenovirus treatment group, which demonstrate that this adenovirus vector inhibit auto-responsive T cells activation and proliferation. Moreover, compared with EAM mice, even administered on days 14-16, the Ad-CIITA treated mice achieved significant reduction in disease severity. It indicates the therapeutic potential of blocking T cells activation by gene-transfer in myocarditis.