Activating mutations of RAS frequently occur in subsets of human cancers, indicating that RAS activation is important for tumorigenesis. However, a large proportion of these cancers still retain wild-type RAS alleles, suggesting that either the RAS pathway is activated in a distinct manner or another pathway is deregulated. To uncover novel tumor-suppressor genes, we screened an RNA-interference library for knockdown constructs that transform human primary cells in the absence of ectopically introduced oncogenic RAS. Here we report the identification of PITX1, whose inhibition induces the RAS pathway and tumorigenicity. Interestingly, we observed low expression of PITX1 in prostate and bladder tumors and in colon cancer cell lines containing wild-type RAS. Restoration of PITX1 in the colon cancer cells inhibited tumorigenicity in a wild-type RAS-dependent manner. Finally, we identified RASAL1, a RAS-GTPase-activating protein, as a transcription target through which PITX1 affects RAS function. Thus, PITX1 suppresses tumorigenicity by downregulating the RAS pathway through RASAL1.