Objectlve To investigate the inhibition of Coxsackievirus B3(CVB3)infection in cardiac myocytes cultured by small interfering RNA(siRNA)-mediated RNA interference and to evaluate the feasibility of siRNA as the prophylaxis and therapy for CVB3 infection.Methods Cardiac myocytes were prepared in vitro and infected with CVB3,and transfected with siRNA by lipofectamin and electroporation.The numbers of beating cardiac myocytes were counted under the microscope.Neutral red staining was used to evaluate the mortality of cardiac myocytes.Antiviral activities of these siRNAs were estimated by observing cytopathic effect(CPE),plaque reduction assay,Western blot assay and RT-PCR.Results siRNA-3753,which aimed at sequence in 2B section of CVB3 genome,displayed a stronger inhibition of CVB3 infection through screening in HeLa cells,siRNA-3753,chosen to transfect cultured neonatal mice cardiac myocytes,Wag observed to keep a good states of growing and beating at 24 h after CVB3 infection.Whereas the cytopathic signature of controlled cells became stopping beating,round and finally the cell fell off the culture plate.The results showed that siRNA-3753 could protect cells significantly,98.1%inhibition of CVB3 replication with electroporation transfection and 78.2%inhibition of CVB3 with liposome transfection.Transfection efficiencies were 56.0 3%and 9.0%by electroporation and lipofectamin,respectively.Conclusion siRNA,which aims at sequence in 2B section of CVB3 genome,can inhibit CVB3 infection in cultured cardiac myocytes.