There is now increasing evidence which suggests an important role for reactive oxygen species (ROS) in the pathogenesis of osteoporosis. However, little is known on the molecular components of the oxidative stress pathway or their functions in bone. In this study, we evaluated the role and mechanism of action of glutaredoxin (Grx) 5, a protein that is highly expressed in bone. Osteoblasts were transfected with Grx5 siRNA and treated with hydrogen peroxide (H(2)O(2)). Grx5 siRNA treatment increased apoptosis while Grx5 overexpression protected MC3T3-E1 cells against H(2)O(2) induced apoptosis and ROS formation. Grx5 deficiency results in impaired biogenesis of Fe-S cluster in yeast. Accordingly, activity of mitochondrial aconitase, whose activity is dependent on Fe-S cluster, decreased in Grx5 siRNA treated cells. Since reduced formation of Fe-S cluster would lead to increased level of free iron, a competitive inhibitor of manganese superoxide dismutase (MnSOD), we measured MnSOD activity in Grx5 deficient osteoblasts and found MnSOD activity was significantly reduced. The consequence of long term inhibition of Grx5 on osteoblast apoptosis was evaluated using lentiviral shRNA technology. Grx5 shRNA cells exhibited higher caspase activity and cardiolipin oxidation in the presence of H(2)O(2). MnSOD activity was rescued by the addition of MnCl(2) to Grx5 shRNA osteoblasts in the presence of H(2)O(2). Our findings are consistent with the hypothesis that Grx5 is an important determinant of osteoblast apoptosis and acts via a molecular pathway that involves regulation of ROS production, cardiolipin oxidation, caspase activity, Fe-S cluster formation, and MnSOD activity.