<p>Phospholipase Cε (PLCε) has been shown to augment inflammation and inflammation-associated carcinogenesis by inducing proinflammatory cytokine expression through activation of nuclear factor-κB (NF-κB), making it a candidate molecular target for development of anti-inflammatory and cancer-preventive agents. Aiming at developing its selective inhibitor, we carry out a high-throughput screening of 68,114 compounds by using a fluorogenic substrate and discover zinc pyrithione (ZPT) as an ε class-specific PLC inhibitor whose IC₅₀ value for phosphatidylinositol 4,5-bisphosphate is 7.5 µM. Further experiments show that Zn²⁺ ion is the active principle of ZPT while the pyrithione moiety acts as an ionophore to raise the intracellular Zn²⁺ concentration. Treatment with ZPT effectively inhibits activation of protein kinase D, nuclear entry of NF-κB and expression of proinflammatory molecules induced by lysophosphatidic acid in cultured colon epithelial cells. Moreover, administration of ZPT not only ameliorates dextran sulfate-induced inflammatory colitis but also inhibits malignant progression of intestinal tumours formed in <i>Apc</i>^<i>Min/+</i> mice. Furthermore, it displays growth-inhibitory and anti-metastatic activities toward xenografts of human colorectal cancer cells. These results support the notion that PLCε inhibitors may become promising anti-inflammatory, cancer-preventive and anti-cancer agents, and suggest that PLCε-inhibition may account for a molecular mechanism underlying the inflammation-moderating activity of zinc.</p>