AbstractManganese (Mn) is an essential micronutrient for plant growth but becomes toxic when present in excess. A number ofArabidopsisproteins are involved in Mn transport including ECA3, MTPs, and NRAMPs; however, their relative contributions to Mn homeostasis remain to be demonstrated. A major focus here was to clarify the importance of ECA3 in responding to Mn deficiency and toxicity using a range of mutants. We show that ECA3 localizes to thetrans‐Golgi and plays a major role in response to Mn deficiency with severe effects seen ineca3 nramp1 nramp2under low Mn supply. ECA3 plays a minor role in Mn‐toxicity tolerance, but only when thecis‐Golgi‐localized MTP11 is non‐functional. We also use mutants and overexpressors to determine the relative contributions of MTP members to Mn homeostasis. Thetrans‐Golgi‐localized MTP10 plays a role in Mn‐toxicity tolerance, but this is only revealed in mutants when MTP8 and MTP11 are non‐functional and when overexpressed inmtp11mutants. MTP8 and MTP10 confer greater Mn‐toxicity resistance to thepmr1yeast mutant than MTP11, and an important role for the first aspartate in the fifth transmembrane domain DxxxD motif is demonstrated. Overall, new insight into the relative influence of key transporters in Mn homeostasis is provided.