A hydroponic experiment with barley seedlings (Hordeum vulgare L. cv. Minorimugi) grown in iron (Fe)-depleted medium in the presence of added arsenic (As) at the rates of 0, 0.67, 6.7 and 67 µmol L−1 (equivalent to 0, 0.05, 0.5 and 5 mg L−1 As, respectively) showed that increasing the As concentration in the medium lowered the release of phytosiderophores (PS) and their concentration in the roots. This Fe-depleted experiment was conducted to clarify the effect of As on the release and concentration of PS in roots and on the phosphorus (P) and Fe concentrations in plants. The chlorophyll index increased substantially in the 67 µmol L−1 As treatment compared with the other treatments. This result indicated that higher concentrations of As might interrupt the appearance of Fe chlorosis in plants grown in Fe-depleted medium. Arsenic at a level of 67 µmol L−1 increased the Fe concentration and accumulation in shoots disappearing the whitish chlorosis. An increased concentration of Fe in the shoot might also be responsible for lowering the release and concentration of PS in the roots. Increases in the concentration of Fe in the shoot most likely resulted from enhanced Fe translocation from the roots to the shoots. The physiological mechanism of the higher Fe translocation with As needs to be investigated. Arsenic lowered the concentrations of P, potassium (K), calcium (Ca) and magnesium (Mg) in the shoot at the 67 µmol L−1 level. A higher Fe concentration and higher ratio of Fe/P in the shoot might be responsible for the greening of the leaves in the 67 µmol L−1 As treatment. The concentrations of manganese (Mn), zinc (Zn) and copper (Cu) were reduced by the high As levels. There was a concomitant increase in the As contents in shoots with higher As levels in the growth medium. The relationship between the concentrations of As and Fe and between P and Fe in the shoots was the opposite. Thus, higher As level might play a role in increasing the mobility of root Fe in barley tissues grown in Fe-depleted medium. Grasses grown under Fe-deficient conditions might not show Fe chlorosis in the presence of high concentrations of As.