© The Royal Society of Chemistry. The increasing levels of heavy metals derived from human activity are poisoning marine environments, threating zooplankton and ocean food webs. To protect themselves from the harmful effects of heavy metals, living beings have different physiological mechanisms, one of which is based on metallothioneins (MTs), a group of small cysteine-rich proteins that can bind heavy metals counteracting their toxicity. The MT system of urochordate appendicularians, an ecologically relevant component of the zooplankton, remained, however, unknown. In this work, we have characterized the MTs of the appendicularian species Oikopleura dioica, revealing that O. dioica has two MT genes, named OdMT1 and OdMT2, which encode for Cys-rich proteins, the former with 72 amino acids comparable with the small size MTs of other organisms, but the second with 399 amino acids representing the longest MT reported to date for any living being. Sequence analysis revealed that OdMT2 gene arose from a duplication of an ancestral OdMT1 gene followed by up to six tandem duplications of an ancestral repeat unit (RU) in the current OdMT2 gene. Interestingly, each RU contained, in turn, an internal repeat of a 7-Cys subunit (X3CX3CX2CX2CX3-6CX2CXCX), which is repeated up to 12 times in OdMT2. Finally, ICP-AES analyses of heterologously expressed OdMT proteins showed that both MTs were capable to form metal-complexes, with preference for cadmium ions. Collectively, our results provide the first characterization of the MT system in an appendicularian species as an initial step to understand the zooplankton response to metal toxicity and other environmental stress situations.