BACKGROUND: The pathogenic Yersinia species exhibit a primarily extracellular lifestyle through manipulation of host signaling pathways that regulate pro-inflammatory gene expression and cytokine release. To identify host genes that are targeted by Yersinia during the infection process, we performed an RNA interference (RNAi) screen based on recovery of host NF-?B-mediated gene activation in response to TNF-a stimulation upon Y. enterocolitica infection. RESULTS: We screened shRNAs against 782 genes in the human kinome and 26 heat shock genes, and identified 19 genes that exhibited = 40% relative increase in NF-?B reporter gene activity. The identified genes function in multiple cellular processes including MAP and ERK signaling pathways, ion channel activity, and regulation of cell growth. Pre-treatment with small molecule inhibitors specific for the screen hits c-KIT and CKII recovered NF-?B gene activation and/or pro-inflammatory TNF-a cytokine release in multiple cell types, in response to either Y. enterocolitica or Y. pestis infection. CONCLUSIONS: We demonstrate that pathogenic Yersinia exploits c-KIT signaling in a T3SS-dependent manner to downregulate expression of transcription factors EGR1 and RelA/p65, and pro-inflammatory cytokines. This study is the first major functional genomics RNAi screen to elucidate virulence mechanisms of a pathogen that is primarily dependent on extracellular-directed immunomodulation of host signaling pathways for suppression of host immunity.