The aim of this study was to investigate the effect of costimulation blockade with cytotoxic T-lymphocyte-associated-antigen 4-immunoglobulin (CTLA4Ig) and anti-CD40L monoclonal antibody (anti-CD40L mAb) on an experimental autoimmune myocarditis (EAM) mouse model. Characteristics of myocardial tissue were observed by hematoxylin and eosin (H&E) staining. The messenger RNA (mRNA) levels of CTLA4, CD40L, IFN-gamma, and IL-4 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Serum concentrations of IFN-gamma and IL-4 were determined by ELISA. After immune intervention, the inflammatory score, mRNA levels of CTLA4 and CD40L, and IFN-gamma level were decreased. Furthermore, these parameters in the combinational intervention group (blockade by CTLA4Ig and anti-CD40L mAb) were significantly decreased, compared to the single intervention group (blockade by CTLA4Ig or anti-CD40L mAb). However, after costimulation, blockade serum IL-4 levels were increased. Therefore, costimulation blockade by combination CTLA4Ig and anti-CD40L mAb could more effectively inhibit the inflammatory response of EAM than single use of CTLA4Ig or anti-CD40L mAb.