Background: Micro-RNAs (miRNAs) are small non-coding RNAs that modulate many target genes. Viral myocarditis is common cardiomyopathy, however, there is an absence of effective therapeutic strategies for viral myocarditis (VMC). The purpose of this research was to characterize changes in miRNAs expression in VMC mice. Methods: Atrial myocytes were infected coxsackievirus B3 and miRNAs microarray was performed. miRNAs target predicted and the bioinformatics analysis was carried out by gene ontology (GO) and KEGG pathway analysis. To validate the results, Difference miRNAswere identified in heart of mice by real-time polymerase chain reaction (PCR). Results: We identified 94 miRNAs that were differentially expressed (27 were up-regulated and 67 were downregulated by at least 2.0-fold). Real time PCR analysis has confirmed that the expression levels of 7 miRNAs upregulated, 18 miRNAs down-regulated. They were mainly involved in protein binding, small GTPase mediated signal transduction, protein phosphorylation by GO. Pathway analysis showed that a significant enrichment in several pathways related to cAMP signaling pathway, AMPK signaling pathway, RAS signaling pathway, Rap1 signaling pathway, ErbB signaling pathway, Oxytocin signaling pathway. Conclusions: Our results provide a better understanding of the mechanisms of viral myocarditis pathophysiology.