The animal pappalysin metalloproteinases, PAPP-A and PAPP-A2, are highly specific regulatory enzymes of the insulin-like growth factor (IGF) system. Cleavage of their only known substrates, a subset of IGF binding proteins (IGFBPs), releases bioactive IGFI and IGFII, thus promoting IGF signaling. Stanniocalcin-1 and -2 (STC1 and STC2) are potent pappalysin inhibitors, completing the STC-PAPP-A-IGFBP-IGF axis. Utilizing homology searches and phylogenetic analyses, we examined the occurrence of pappalysins in the animal kingdom and their functional conservation. This revealed the extensive presence of pappalysins across metazoans, as well as the presence of 3 pappalysins: PAPP-A, PAPP-A2, and a third group of invertebrate pappalysins, which we name invertebrate PAPP-A (invPAPP-A). We show that PAPP-A and PAPP-A2 arose by duplication during early vertebrate evolution. Despite significant evolutionary distance, the domain architecture of the metazoan pappalysins is completely conserved, and several functional domains and motifs are highly conserved across all pappalysins. However, invPAPP-A exists outside the context of IGFBPs, suggesting that the animal pappalysins may have substrates beyond the IGFBPs for PAPP-A and PAPP-A2 that remain to be discovered. Since PAPP-A is an emerging drug target, it is important to understand potential involvement in regulatory systems other than the IGF system, which might be affected upon targeting of PAPP-A.