Pathogenic Yersinia contain a virulence plasmid that encodes genes for intracellular effectors, which neutralize the host immune response. One effector, YopM, is necessary for Yersinia virulence, but its function in host cells is unknown. To identify potential cellular pathways affected by YopM, proteins that co-immunoprecipitate with YopM in mammalian cells were isolated and identified by mass spectrometry. Results demonstrate that two kinases, protein kinase C-like 2 (PRK2) and ribosomal S6 protein kinase 1 (RSK1), interact directly with YopM. These two kinases associate only when YopM is present, and expression of YopM in cells stimulates the activity of both kinases. RSK1 is activated directly by interaction with YopM, and RSK1 kinase activity is required for YopM-stimulated PRK2 activity. YopM activation of RSK1 occurs independently of the actions of YopJ on the MAPK pathway. YopM is also required for Yersinia-induced changes in RSK1 mobility in infected macrophage cells. These results identify the first intracellular targets of YopM and suggest YopM acts to stimulate the activity of PRK2 and RSK1.