An ancient evolutionary innovation of a novel cell-type, the stinging cell (cnidocyte), appeared >600 million years ago in the phylum Cnidaria (sea anemones, corals, hydroids, and jellyfish). A complex bursting nano-injector of venom, the cnidocyst, is embedded in cnidocytes and enables cnidarians paralyzing prey and predators, contributing to the evolutionary success of this phylum. In this work, we show that post-transcriptional regulation by a pan-cnidarian microRNA, miR-2022, is essential for biogenesis of these cells. By manipulation of miR-2022 levels in a transgenic reporter line of cnidocytes in the sea anemone Nematostella vectensis, followed by transcriptomics, single-cell data analysis, prey paralysis assays, and cell sorting of transgenic cnidocytes, we reveal that miR-2022 enables cnidocyte biogenesis, while exhibiting a conserved expression domain with its targets in cnidocytes of other cnidarian species. Thus, here we reveal one of the most ancient microRNA-regulated processes in nature by studying the functional basis for its conservation.