The bicistronic microRNA (miRNA) locus miR-144/451 is highly expressed during erythrocyte development, although its physiological roles are poorly understood. We show that miR-144/451 ablation in mice causes mild erythrocyte instability and increased susceptibility to damage after exposure to oxidant drugs. This phenotype is deeply conserved, as miR-451 depletion synergizes with oxidant stress to cause profound anemia in zebrafish embryos. At least some protective activities of miR-451 stem from its ability to directly suppress production of 14-3-3ζ, a phospho-serine/threonine-binding protein that inhibits nuclear accumulation of transcription factor FoxO3, a positive regulator of erythroid anti-oxidant genes. Thus, in miR-144/451-/- erythroblasts, 14-3-3ζ accumulates, causing partial relocalization of FoxO3 from nucleus to cytoplasm with dampening of its transcriptional program, including anti-oxidant-encoding genes Cat and Gpx1. Supporting this mechanism, overexpression of 14-3-3ζ in erythroid cells and fibroblasts inhibits nuclear localization and activity of FoxO3. Moreover, shRNA suppression of 14-3-3ζ protects miR-144/451-/- erythrocytes against peroxide-induced destruction, and restores catalase activity. Our findings define a novel miRNA-regulated pathway that protects erythrocytes against oxidant stress, and, more generally, illustrate how a miRNA can influence gene expression by altering the activity of a key transcription factor. © 2010 by Cold Spring Harbor Laboratory Press.