ABSTRACT Acidocalcisome-like organelles are found in all kingdoms of life. Many of their functions, such as the accumulation and storage of metal ions, nitrogen and phosphate, the activation of blood clotting and inflammation, depend on the controlled synthesis and turnover of polyphosphate (polyP), a polymer of inorganic phosphate linked by phosphoric anhydride bonds. The exploration of the role of acidocalcisomes in metabolism and physiology requires the manipulation of polyP turnover, yet the complete set of proteins responsible for this turnover is unknown. Here, we identify a novel type of polyphosphatase operating in the acidocalcisome-like vacuoles of the yeast Saccharomyces cerevisiae, which we called Ppn2. Ppn2 belongs to the PPP-superfamily of metallophosphatases, is activated by Zn2+ ions and exclusively shows endopolyphosphatase activity. It is sorted to vacuoles via the multivesicular body pathway. Together with Ppn1, Ppn2 is responsible for a substantial fraction of polyphosphatase activity that is necessary to mobilize polyP stores, for example in response to phosphate scarcity. This finding opens the way to manipulating polyP metabolism more profoundly and deciphering its roles in phosphate and energy homeostasis, as well as in signaling.