Protein kinase C iota (PKCiota) has been implicated in Ras signaling, however, a role for PKCiota in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCiota is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCiota in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCiota (kdPKCiota) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCiota in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCiota. Our data demonstrate that PKCiota is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCiota, and Rac1.