Nudt16p is a nuclear RNA decapping protein initially identified in Xenopus (X29) and known to exist in mammals. Here, we identified putative orthologs in 57 different organisms ranging from humans to Cnidaria (anemone/coral). In vitro analysis demonstrated the insect ortholog can bind RNA and hydrolyze the m(7)G cap from the 5'-end of RNAs indicating the Nudt16 gene product is functionally conserved across metazoans. This study also identified a closely related paralogous protein, known as Syndesmos, which resulted from a gene duplication that occurred in the tetrapod lineage near the amniote divergence. While vertebrate Nudt16p is a nuclear RNA decapping protein, Syndesmos is associated with the cytoplasmic membrane in tetrapods. Syndesmos is inactive for RNA decapping but retains RNA-binding activity. This structure/function analysis demonstrates evolutionary conservation of the ancient Nudt16 protein suggesting the existence and maintenance of a nuclear RNA degradation pathway in metazoans.