Effects of adding Fe2+ ions in photoheterotrophic cultivation of Dunaliella salina on growth and β-carotene synthesis were investigated. Different concentrations of Fe2+ ions and organic carbon source (acetate and malonate) were studied. A significant increase of cellular β-carotene content was observed, with a maximum value of 70 pg/cell in a culture enriched with 67.5 mM of acetate and 450 μM of FeSO4 and 33 pg/cell in the case of 67.5 mM of malonate and 450 μM of FeSO4. For comparison, the well-known nitrate starvation protocol was applied, and 35 pg/cell of β-carotene were obtained. By inducing an oxidative stress, Fe2+ ions revealed to stimulate β-carotene synthesis, especially when acting in the presence of the carbon source. Furthermore for some concentration of Fe2+ ions and organic carbon source, the loss in cells division was reduced compared to nitrate starvation.