Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and their presence has also been confirmed in several bacterial species. Recently, we reported for the first time in silico evidence of RIP encoding genes in metazoans, in two closely related species of insects: Aedes aegypti and Culex quinquefasciatus. Here, we have experimentally confirmed the presence of these genes in mosquitoes and attempted to unveil their evolutionary history. A detailed study was conducted, including evaluation of taxonomic distribution, phylogenetic inferences and microsynteny analyses, indicating that the culicine RIP genes derived from a single Horizontal Gene Transfer (HGT) event, probably from a Cyanobacterial donor species. Moreover, evolutionary analyses show that, after transference, these genes evolved under purifying selection, strongly suggesting that they play functional roles in these organisms. In this work we confirm the presence of RIP genes in Culicinae species, and show solid evidence supporting the hypothesis that these genes are derived from a single prokaryotic transferred gene through HGT. In addition, clear evidence of purifying selection pressure has been recorded, supporting the hypothesis that these genes are functional within this subfamily.