Type III secretion systems are used by several pathogens to translocate effector proteins into host cells. Yersinia pseudotuberculosis delivers several Yop effectors (e.g. YopH, YopE and YopJ) to counteract signalling responses during infection. YopB, YopD and LcrV are components of the translocation machinery. Here, we demonstrate that a type III translocation protein stimulates proinflammatory signalling in host cells, and that multiple effector Yops counteract this response. To examine proinflammatory signalling by the type III translocation machinery, HeLa cells infected with wild-type or YopY. pseudotuberculosis strains were assayed for interleukin (IL)-8 production. HeLa cells infected with a YopEHJ triple mutant released significantly more IL-8 than HeLa cells infected with isogenic wild-type, YopE, YopH or YopJ bacteria. Complementation analysis demonstrated that YopE, YopH or YopJ are sufficient to counteract IL-8 production. IL-8 production required YopB, but did not require YopD, pore formation or invasin-mediated adhesion. In addition, YopB was required for activation of nuclear factor kappa B, the mitogen-activated protein kinases ERK and JNK and the small GTPase Ras in HeLa cells infected with the YopEHJ mutant. We conclude that interaction of the Yersinia type III translocator factor YopB with the host cell triggers a proinflammatory signalling response that is counteracted by multiple effectors in host cells.