Liposome-mediated gene delivery provides a powerful strategy for the study of gene function and for gene therapy. Coxsackievirus B3 is an important human pathogen associated with various diseases. Here we reported that liposome-mediated transient transfection of plasmid cDNA inhibited coxsackieviral replication at the levels of RNA, protein and viral progeny release. These inhibitory effects were observed in various cell types and by using different liposome reagents. We further showed that the inhibition was likely due to the lack of virus attachment. Moreover, we showed that addition of cholesterol restored, at least in part, the viral infectivity. Interestingly, we found that membrane cholesterol levels were unchanged during transfection, indicating that disruption rather than depletion of membrane cholesterol contributes to the inhibitory effects of transfection. Our data suggest that liposome-mediated cDNA transient transfection inhibits coxsackievirus infectivity via inhibition of viral attachment, which is likely occurring through the changes of membrane cholesterol integrity.