Phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (pChk2) induced by DNA double-strand breaks is required for inhibition of cell cycle progression in the G2 phase. The purpose of the present paper was to investigate the expression of wild-type p53-induced phosphatase 1 (Wip1 or PPM1D), a negative regulator of Chk2, to better understand its role in human gastric cancer. In non-neoplastic gastric mucosa, most epithelial cells exhibited Wip1-positive and pChk2-negative immunoreactivity, whereas an inverse pattern of protein expression was detected at the surface of the foveolar epithelium. In tumor tissues, 74% of 53 gastric cancers had intense Wip1 immunoreactivity and close correlation with both tumor size (P = 0.0497) and Chk2 dephosphorylation (P = 0.0213). In MKN-74 gastric cancer cells, ionizing radiation (IR)-induced Wip1 upregulation was detected at protein levels, but the Chk2-mediated cell cycle regulatory mechanism was disrupted. In addition, protease inhibitor Z-Leu-Leu-Leu (ZLLL) effectively upregulated Wip1 levels in the presence or absence of IR, suggesting that Wip1 expression can be modulated post-transcriptionally. Understanding the Wip1-mediated signaling pathway in gastric cancer may provide useful information for the development of new chemo- and radiotherapies.