Peroxynitrite crosses the red blood cell (RBC) membrane and reacts with hemoglobin (Hb) producing mainly metHb, which is reduced back to ferrousHb by NADH- and NADPH-dependent reductases. Peroxynitrite also induces band 3 (B3) tyrosine phosphorylation, a signaling pathway believed to activate glucose metabolism. This study was aimed to decipher the relationship between these two peroxynitrite-dependent processes. Peroxynitrite induced a burst of the hexose monophosphate shunt (HMS), revealed by NMR studies, and a burst of the glycolytic pathway, measured by lactate production. The HMS plays a prominent role in membrane signaling, as demonstrated by B3 phosphotyrosine inhibition by the glycolytic pathway inhibitor 2-deoxy-glucose (2DG) and activation by dehydroepiandrosterone (DHEA), an inhibitor of HMS. Peroxynitrite-induced B3 tyrosine phosphorylation was paralleled by the inhibition of membrane-associated phosphotyrosine phosphatase (PTP) activity, which was protected by 2DG but not DHEA. Interestingly, heme poisoning with CO inhibited peroxynitrite-dependent Hb oxidation and lactate production but did not affect PTP down regulation. These results suggest two distinct and concurrent effects of peroxynitrite: one mediated by Hb which, likely in its oxidized state, binds more strongly to B3, and another mediated by PTP-dependent B3 phosphorylation. Both effects are directed towards a surge in glucose utilization.