Arsenic trioxide (As2O3) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As2O3 induces phosphorylation/activation of the mitogen- activated protein kinase signal-integrating kinases (Mnk) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38 Map kinase gene, indicating that it requires upstream engagement of the p38 Map kinase pathway. Studies using Mnk1-/- or Mnk2 -/-, or double Mnk1-/-Mnk2-/- knockout cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E (eIF4E) at Ser209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide-responses. Consistent with this, pharmacological inhibition of Mnk-activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.