In addition to mediating regulation of endogenous gene expression, RNA interference (RNAi) in plants and invertebrates plays a crucial role in defense against viruses via virus-specific siRNAs. Different studies have demonstrated that the functional diversity of RNAi in animals is linked to the diversification of the Argonaute superfamily, central components of RISCs (RNA induced silencing complexes). The animal Argonaute superfamily is traditionally grouped into AGO and PIWI Argonautes. Yet, by performing phylogenetic analyses and determining the selective evolutionary pressure in the metazoan Argonaute superfamily, we provide evidence for the existence of three conserved Argonaute lineages between basal metazoans and protostomes, namely siRNA-class AGO, miRNA-class AGO and PIWI Argonautes. In addition, it shown that the siRNA-class AGO lineage is characterized by high rates of molecular evolution, suggesting a role in the arms race with viruses, while the miRNA-class AGOs display strong sequence conservation. Interestingly, we also demonstrate that vertebrates lack siRNA-class AGO proteins and that vertebrate AGOs display low rates of molecular evolution. In this way, we provide supportive evidence for the loss of the antiviral siRNA-class AGO group in vertebrates and discuss the consequence hereof on antiviral immunity and the use of RNAi as a loss of function tool in these animals.