Coxsackievirus B3 (CVB3) is a principal causative agent of viral myocarditis, meningitis and pancreatitis. There is no vaccine available for clinical use. It has been demonstrated that the primary molecular determinant of virulence phenotype is located in the 5' UTR of the viral genome. Translation initiation of CVB3 RNA is directed by the IRES element situated in the 5'UTR. In the present study, we analyse the effects of single point mutations introduced in different positions in the domain V of the IRES RNA of CVB3 wild type. We characterize in vitro virus replicative capacitiy and translation efficiency and we test in vivo virulence of different CVB3 mutants produced by the introduction of different mutations in the domain V of IRES by site-directed mutagenesis to abolish its structure. Our results demonstrate that all RNA mutants display different levels of decreased replication and translation initiation efficiency in vitro. The translation defect was correlated with significant reduced viral titer of mutant particles in comparison with the wild type. When inoculated in mice, mutant viruses were checked for inflammation and necrosis.In vitro and in vivo Findings strongly suggest that the most attenuated mutant strain could be considered a candidate for live-attenuated CVB3 vaccine.