Background: The serine/threonine kinase IKBKE is frequently overexpressed or activated in a variety of human cancers. Ectopic expression of IKBKE induces malignant transformation, cell migration, invasion and chemoresistance. Thus, IKBKE is an attractive target for anti-cancer drug development. Methods: By screening of NCI Diversity Set and Clinical Collection I and II compound libraries using cell-based assay, we identified several candidates of IKBKE inhibitors, which directly inhibited IKBKE kinase activity in vitro and in vivo. One of them, malachite green oxalate (MCCK1), was further characterized. The mechanism was examined by western blot, immunoprecipitation (IP) and Immunofluorescence. We also evaluated in a mouse xenograft model. In vitro kinase assay and luciferase reporter assay were also performed in our experiments. Results: MCCK1 inhibits IKBKE kinase as well as its downstream targets such as IkBa, p65 and IRF3. MCCK1 is a selective inhibitor for IKBKE, with moderate effect on TBK1, but does not inhibit the activation of IKK alpha/beta, STAT3, Erk-1/2, p38 or JNK. The inhibition of IKBKE by MCCK1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor aberrant expression of IKBKE. Furthermore, MCCK1 inhibits tumor growth in nude mice of human cancer cells in which IKBKE is elevated but not of those cancer cells in which it is not. Conclusion: These data indicate that MCCK1 is an IKBKE inhibitor with anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients with tumors over expressing IKBKE.