The bacterial pathogen Salmonella typhimurium triggers host cell signaling pathways that lead to cytoskeletal and nuclear responses required for pathogenesis. Here, the role of the small guanosine triphosphate (GTP)-binding protein CDC42Hs in these responses was examined. Expression of a dominant interfering mutant of CDC42 (CDC42HsN17) prevented S. typhimurium-induced cytoskeletal reorganization and subsequent macropinocytosis and bacterial internalization into host cells. Cells expressing constitutively active CDC42 (CDC42HsV12) internalized an S. typhimurium mutant unable to trigger host cell responses. Furthermore, expression of CDC42HsN17 prevented S. typhimurium-induced JNK kinase activation. These results indicate that CDC42 is required for bacterial invasion and induction of nuclear responses in host cells.