Plant vacuoles are the main cellular reservoirs to store K+ . The vacuolar K+ channels play a pivotal role in K+ exchange between cytosol and vacuolar sap. Among vacuolar K+ transporters, the Two Pore Potassium Channels (TPKs) are highly selective K+ channels present in most or all plant vacuoles and could be involved in various plant stress responses and developmental processes. Although the majority of TPK members have a vacuolar specialisation, some TPKs display different membrane localisation including the plasma membrane, tonoplast of protein storage vacuoles and probably chloroplast membranes. The functional properties as well as physiological roles of TPKs remains largely unexplored. In this review, we have collected recent data about the physiology, structure, functionality and evolution of TPK/KCO3 channels. We also critically evaluate the latest findings on the biological role, physiological functions, and regulation of TPK/KCO3 channels in relation to their structure and phylogenetic position. The possible role of TPK/KCO3 channels in plant tolerance to various abiotic stresses is summarised, and the future priority directions for TPK/KCO3 studies are addressed.