The interplay between pathogen-encoded virulence factors and host cell signaling networks is critical for both the establishment and clearance of microbial infections. Yersinia uptake into host cells serves as an in vitro model for exploring how host cells respond to Yersinia adherence. In this study, we provide insight into the molecular nature and regulation of signaling networks that contribute to the uptake process. Using a reconstitution approach in Fak(-/-) fibroblasts, we have been able to specifically address the interplay between Fak, Cas and Pyk2 in this process. We show that both Fak and Cas play roles in the Yersinia uptake process and that Cas can function in a novel pathway that is independent of Fak. Fak-dependent Yersinia uptake does not appear to involve Cas-Crk signaling. By contrast, Cas-mediated uptake in the absence of Fak requires Crk as well as the protein tyrosine kinases Pyk2 and Src. In spite of these differences, the requirement for Rac1 activity is a common feature of both pathways. Furthermore, blocking the function of either Fak or Cas induces similar morphological defects in Yersinia internalization, which are manifested by incomplete membrane protrusive activity that is consistent with an inhibition of Rac1 activity. Pyk2 also functions in Yersinia uptake by macrophages, which are physiologically important for clearing Yersinia infections. Taken together, these data provide new insight into the host cellular signaling networks that are initiated upon infection with Y. pseudotuberculosis. Importantly, these findings also contribute to a better understanding of other cellular processes that involve actin remodeling, including the host response to other microbial pathogens, cell adhesion and migration.