Iron deficiency is observed in nearly half of the heart failure patients whilst closely correlated with mitochondrial dysfunction. Besides the structural roles in mitochondria, iron is also the cofactor of the hypoxia inducible factor (Hif) degradating enzyme, prolylhydroxylase, thereby Hif accumulation and its related metabolic effects commonly involve in iron deficiency. In this study, we used atrium derived HL-1 cells to investigate the effects of iron depletion on mitochondrial function under in vitro conditions. We aimed to discriminate the Hif dependent effects of iron deprivation on mitochondrial function to reveal the mechanisms leading to cardiac failure. For this purpose, HL-1 cells were either directly incubated with the iron chelating agent deferoxamine (DFO) or with dimethyloxalylglycine (DMOG, inhibitor of prolylhydroxylase). Mitochondrial function was evaluated by measuring cellular ATP content and mitochondrial potential (psi). According to our results, 48 h of DFO incubation affected cell viability and ATP production through further mechanisms additional to Hif-1 alpha accumulation. Unlike DMOG group, DFO incubation did not disturb mitochondrial function probably due to its low permeability. Whether or not, prolyl hydroxylase inhibition without iron depletion may negatively affect mitochondrial function through Hif dependent mechanisms.