Glucose-6-phosphate dehydrogenase (G6PD) plays a key role in the regeneration of NADPH and maintenance of cellular redox balance. In the present study, we investigate the effect of G6PD deficiency on H₂O₂-elicited signaling in HepG2 cells. H₂O₂ was found to inhibit cellular protein tyrosine phosphatase (PTP) activity, resulting in activation of MAPKs. MKP-1 expression increased in the late phase of H₂O₂ signaling. Using RNAi technology, we found that G6PD knockdown enhanced the inhibitory effect of H₂O₂ on PTPs and led to sustained MAPK activation. This was accompanied by delayed expression and inhibition of MKP-1. Using a pharmacological inhibitor and siRNA, we demonstrate that MKP-1 acts as a regulator of MAPK activation in H₂O₂ signaling. The prolonged MAPK activation in G6PD-knockdown cells was associated with an increased susceptibility to H₂O₂-induced apoptosis and growth retardation. Treatment with p38 and JNK inhibitors or N-acetylcysteine ameliorated such cellular effect, while triptolide and MKP-1-siRNA did the opposite. Glucose oxidase treatment had similar effects as addition of H₂O₂. Taken together, these findings suggest that G6PD knockdown enhances the magnitude and duration of H₂O₂-induced MAPK signaling through inhibition of cellular PTPs, and the resultant anomalous signaling may lead to cell demise. © 2010 Elsevier Inc.