Flooding of soils results in acute oxygen deprivation (anoxia) of plant roots during winter in temperate latitudes, or after irrigation, and is a major problem for agriculture. One early response of plants to anoxia and other environmental stresses is downregulation of water uptake due to inhibition of the water permeability (hydraulic conductivity) of roots (Lp(r)). Root water uptake is mediated largely by water channel proteins (aquaporins) of the plasma membrane intrinsic protein (PIP) subgroup. These aquaporins may mediate stress-induced inhibition of Lp(r) but the mechanisms involved are unknown. Here we delineate the whole-root and cell bases for inhibition of water uptake by anoxia and link them to cytosol acidosis. We also uncover a molecular mechanism for aquaporin gating by cytosolic pH. Because it is conserved in all PIPs, this mechanism provides a basis for explaining the inhibition of Lp(r) by anoxia and possibly other stresses. More generally, our work opens new routes to explore pH-dependent cell signalling processes leading to regulation of water transport in plant tissues or in animal epithelia.