Manganese (Mn) is an essential element, acting as cofactor in numerous enzymes. In particular, a Mn cluster is indispensable for the function of the oxygen-evolving complex of photosystem II. Metal transporters of the Natural Resistance-Associated Macrophage Protein (NRAMP) family have the ability to transport both iron and Mn. AtNRAMP3 and AtNRAMP4 are required for iron mobilization in germinating seeds. The results reported here show that, in adult Arabidopsis (Arabidopsis thaliana) plants, AtNRAMP3 and AtNRAMP4 have an important role in Mn homeostasis. Vacuolar Mn accumulation in mesophyll cells of rosette leaves of adult nramp3nramp4 double mutant plants was dramatically increased when compared with the wild type. This suggests that a considerable proportion of the cellular Mn pool passes through the vacuole and is retrieved in an AtNRAMP3/AtNRAMP4-dependent manner. The impaired Mn release from mesophyll vacuoles of nramp3nramp4 double mutant plants is associated with reduced growth under Mn deficiency. However, leaf AtNRAMP3 and AtNRAMP4 protein levels are unaffected by Mn supply. Under Mn deficiency, nramp3nramp4 plants contain less functional photosystem II than the wild type. These data are consistent with a shortage of Mn to produce functional photosystem II, whereas mitochondrial Mn-dependent superoxide dismutase activity is maintained under Mn deficiency in both genotypes. The results presented here suggest an important role for AtNRAMP3/AtNRAMP4-dependent Mn transit through the vacuole prior to the import into chloroplasts of mesophyll cells.