Pathogenic Yersinia species employ type III machines to secrete YopBDR into the extracellular milieu. After attaching to host cells, yersiniae transform the type III machinery into an injection device and target YopEHMNOPT into eukaryotic cells. Yersinia pseudotuberculosis LcrQ is a transcriptional regulator that prevents the expression of yop genes. We report that LcrQ is injected into eukaryotic cells. YscM1, the transciptional regulator of Yersinia enterocolitica, is also injected into eukaryotic cells, whereas the related YscM2 protein remains associated with bacterial cells. Type III targeting of YscM1 requires binding to the SycH chaperone. Chaperone binding as well as depletion of YscM1 and YscM2 from the cytoplasm of Y. enterocolitica causes an increase in yop expression, whereas a block in regulator export reduces expression. We propose a model whereby the chaperone-mediated injection of LcrQ/YscM1 functions as a regulatory switch for bacteria that are attached to host cells, triggering the expression of Yops that travel the type III targeting pathway.