The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-κB kinase β (IKKβ) and confirmed with RNA interference toward IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the nine-gene ovarian cancer-specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion, and adhesion. Downregulating IKKβ activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity. ©2010 AACR.