Summary Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject bacterial effector proteins directly into the host cytosol. One of these effectors, the Yersinia serine/threonine protein kinase YpkA, is an essential virulence determinant involved in host actin cytoskeletal rearrangements and in inhibition of phagocytosis. Here we report that YpkA inhibits multiple Gαq signaling pathways. The kinase activity of YpkA is required for Gαq inhibition. YpkA phosphorylates Ser47, a key residue located in the highly conserved diphosphate binding loop of the GTPase fold of Gαq. YpkA-mediated phosphorylation of Ser47 impairs guanine nucleotide binding by Gαq. Y. pseudotuberculosis expressing wild-type YpkA, but not a catalytically inactive YpkA mutant, interferes with Gαq-mediated signaling pathways. Identification of a YpkA-mediated phosphorylation site in Gαq sheds light on the contribution of the kinase activity of YpkA to Yersinia pathogenesis.