The effect of additional iron (Fe) on arsenic (As) induced chlorosis in barley (Hordeum vulgare L. cv. Minorimugi) was investigated. The treatments were: (1) 0 μmol L−1 As + 10 μmol L−1 Fe3+ (control), (2) 33.5 μmol L−1 As + 10 μmol L−1 Fe3+ (As-treated) and (3) 33.5 μmol L−1 As + 50 μmol L−1 Fe3+ (additional-Fe3+) for 14 days. Arsenic and Fe3+ were added as sodium-meta arsenite (NaAsO2) and ethylenediaminetetraacetic acid-Fe3+, respectively. Chlorosis in fully developed young leaves was observed in the As-treated plants. The chlorophyll index and the Fe concentration decreased in shoots of the As-treated plants compared with the control plants. Arsenic reduced the concentration of phosphorus, potassium, calcium, magnesium, manganese, zinc and copper. The additional-Fe3+ treatment increased the chlorophyll index in plants compared with the As-treated plants. Among the elements, Fe concentration and accumulation specifically increased in the shoots of additional-Fe3+ plants compared with As-treated plants, indicating that As-induced chlorosis was Fe-chlorosis. Arsenic and Fe were mostly concentrated in the roots of the As-treated plants. Despite inducing chlorosis in the As-treated plants, phytosiderophores (PS) accumulation in the roots and release from the roots did not increase, rather PS accumulation decreased, indicating that As toxicity hindered PS production in the roots. The PS accumulation in the roots was further reduced in the additional-Fe3+ treatment.