The availability of iron for roots has been demonstrated as a critical factor in plant production. The addition of synthetic iron chelates to soil is a common practice in agriculture, which is not economically beneficial. Besides, chemical iron fertilizers cause many problems such as food contamination and environmental pollution. Development of natural Ferrioxamine B as an efficient and safe iron source may be the best strategy to overcome plant iron deficiency and prevention of synthetic agent pollution. The present study investigates the ability of a hydroxamate type siderophores (Ferrioxamine B) as a substitute Fe source during tissue culture of chickpea plants. For this purpose, embryo axes from chickpea seeds were surface sterilized and cultured in 1/2MS and MS culture media including 3% sucrose and 0.8% agar with Ferrioxamine B or Fe–EDTA. The root and shoot length, shoot and root dry weight, total fresh and dry weight, as well as chlorophylls a and b were analysed. Results indicated that Ferrioxamine B did not increase chlorophylls a and b in comparison with Fe-EDTA. However, rooting (22%), total dry weight (38%) and root (75%) and shoot (22%) dry weight significantly (p≤0.05) increased in MS containing Ferrioxamine B in comparison with Fe-EDTA. Consequently, Ferrioxamine B is introduced as a cost-effective and applicable Fe source to favour iron deficiency in vitro.