Accumulation of methionine sulfoxide (Met(O)) is a significant feature of human cataract and previous studies have shown that methionine sulfoxide reductase A (MsrA), which acts to repair Met(O), can defend human lens cells against oxidative stress induced cell death. A key feature of oxidative stress is increased reactive oxygen species (ROS) in association with loss of mitochondrial function. Here, we sought to establish a potential role for MsrA in the accumulation of ROS in lens cells and the corresponding mitochondrial membrane potential in these cells. Targeted gene silencing was used to establish populations of lens cells expressing different levels of MsrA, and the mitochondrial membrane potential and ROS levels of these cell populations were monitored. Decreased MsrA levels were found to be associated with loss of cell viability, decreased mitochondrial membrane potential, and increased ROS levels in the absence of oxidative stress. These effects were augmented upon oxidative stress treatment. These results provide evidence that MsrA is a major determinant for accumulation of ROS in lens cells and that increased ROS levels in lens cells are associated with a corresponding decrease in mitochondrial membrane potential that is likely related to the requirement for MsrA in lens cell viability.