Oxygen depletion under waterlogged conditions results in a compromised operation of H+-ATPase, with strong implications for membrane potential maintenance, cytosolic pH homeostasis, and transport of all nutrients across membranes. The above effects, however, are highly tissue specific and time dependent, and the causal link between hypoxia-induced changes to the cell’s ionome and plant adaptive responses to hypoxia is not well established. This work aimed to fill this gap and investigate the effects of oxygen deprivation on K+ signalling and homeostasis in plants, and potential roles of GORK (depolarization-activated outward-rectifying potassium) channels in adaptation to oxygen-deprived conditions in barley. A significant K+ loss was observed in roots exposed to hypoxic conditions; this loss correlated with the cell’s viability. Stress-induced K+ loss was stronger in the root apex immediately after stress onset, but became more pronounced in the root base as the stress progressed. The amount of K+ in shoots of plants grown in waterlogged soil correlated strongly with K+ flux under hypoxia measured in laboratory experiments. Hypoxia induced membrane depolarization; the severity of this depolarization was less pronounced in the tolerant group of cultivars. The expression of GORK was down-regulated by 1.5-fold in mature root but it was up-regulated by 10-fold in the apex after 48 h hypoxia stress. Taken together, our results suggest that the GORK channel plays a central role in K+ retention and signalling under hypoxia stress, and measuring hypoxia-induced K+ fluxes from the mature root zone may be used as a physiological marker to select waterlogging-tolerant varieties in breeding programmes.