Aims: This study was performed to analyse the molecular characteristics of genes encoding for the major virulence factors in Bacillus anthracis vaccine strain `Carbosap' compared with the wild B. anthracis strain, to evaluate the basis of attenuation. Methods and Results: The molecular characteristics of the B. anthracis `Carbosap' vaccine strain, used as vaccine in Italy, were analysed in comparison with a B. anthracis virulent strain. Despite the presence of the two virulence plasmids pXO1 and pXO2, the `Carbosap' strain proved to be protective for cattle. The presence of the regulatory genes atxA and pagR and the gerX operon, known to be involved in the virulence, was verified. In addition, all genes were sequenced. The results showed that no molecular differences between `Carbosap' and the virulent strain were evident. Conclusions: The results of this study indicate that the attenuation of the `Carbosap' vaccine strain is not due to the lack of virulence genes or to modifications occurring on the sequence of these genes. Therefore, other virulence factors, still unknown, could be involved in the pathogenic mechanisms. Significance and Impact of the Study: This paper adds new information regarding the molecular characteristics of the vaccine strain `Carbosap' and highlights the need to better understand the virulence factors involved in the pathogenicity of B. anthracis strains.