Human CISD2 and mitoNEET are two NEET proteins anchored in the endoplasmic reticulum and mitochondria membranes respectively, with an Fe–S containing domain stretching out in the cytosol. Their cytosolic domains are close in sequence and structure. In the present study, combining cellular and biochemical approaches, we compared both proteins in order to possibly identify specific roles and mechanisms of action in the cell. We show that both proteins exhibit a high intrinsic stability and a sensitivity of their cluster to oxygen. In contrast, they differ in according to expression profiles in tissues and intracellular half-life. The stability of their Fe–S cluster and its ability to be transferred in vitro are affected differently by pH variations in a physiological and pathological range for cytosolic pH. Finally, we question a possible role for CISD2 in cellular Fe–S cluster trafficking. In conclusion, our work highlights unexpected major differences in the cellular and biochemical features between these two structurally close NEET proteins.