Metal homeostasis is critical for the survival of living organisms, and metal transporters play central roles in maintaining metal homeostasis in the living cells. We have investigated the function of a metal transporter of the NRAMP family, AtNRAMP3, in Arabidopsis thaliana. A previous study showed that AtNRAMP3 expression is upregulated by iron (Fe) starvation and that AtNRAMP3 protein can transport Fe. In the present study, we used AtNRAMP3 promoter beta-glucoronidase (GUS) fusions to show that AtNRAMP3 is expressed in the vascular bundles of roots, stems, and leaves under Fe-sufficient conditions. This suggests a function in long-distance metal transport within the plant. Under Fe-starvation conditions, the GUS activity driven by the AtNRAMP3 promoter is upregulated without any change in the expression pattern. We analyze the impact of AtNRAMP3 disruption and overexpression on metal accumulation in plants. Under Fe-sufficient conditions, AtNRAMP3 overexpression or disruption does not lead to any change in the plant metal content. Upon Fe starvation, AtNRAMP3 disruption leads to increased accumulation of manganese (Mn) and zinc (Zn) in the roots, whereas AtNRAMP3 overexpression downregulates Mn accumulation. In addition, overexpression of AtNRAMP3 downregulates the expression of the primary Fe uptake transporter IRT1 and of the root ferric chelate reductase FRO2. Expression of AtNRAMP3::GFP fusion protein in onion cells or Arabidopsis protoplasts shows that AtNRAMP3 protein localizes to the vacuolar membrane. To account for the results presented, we propose that AtNRAMP3 influences metal accumulation and IRT1 and FRO2 gene expression by mobilizing vacuolar metal pools to the cytosol.