Abstract Epidermal growth factor receptors (EGFR) contribute to colonic tumorigenesis in experimental models of colon cancer. We previously showed that EGFR was also required for colonic tumor promotion by Western diet. The goal of this study was to identify EGFR-regulated microRNAs that contribute to diet-promoted colonic tumorigenesis. Murine colonic tumors from Egfrwt and hypomorphic Egfrwa2 mice were screened using micro RNA (miRNA) arrays and miR-143 and miR-145 changes confirmed by Northern, real-time PCR, and in situ analysis. Rodent and human sporadic and ulcerative colitis (UC)-associated colon cancers were examined for miR-143 and miR-145. Effects of EGFR on miR-143 and miR-145 expression were assessed in murine and human colonic cells and their putative targets examined in vitro and in vivo. miR-143 and miR-145 were readily detected in normal colonocytes and comparable in Egfrwt and Egfrwa2 mice. These miRNAs were downregulated in azoxymethane and inflammation-associated colonic tumors from Egfrwt mice but upregulated in Egfrwa2 tumors. They were also reduced in human sporadic and UC colon cancers. EGFR signals suppressed miR-143 and miR-145 in human and murine colonic cells. Transfected miR-143 and miR-145 inhibited HCT116 cell growth in vitro and in vivo and downregulated G1 regulators, K-Ras, MYC, CCND2, cdk6, and E2F3, putative or established targets of these miRNAs. miRNA targets Ras and MYC were increased in colonic tumors from Egfrwt but not Egfrwa2 mice fed a Western diet. EGFR suppresses miR-143 and miR-145 in murine models of colon cancer. Furthermore, Western diet unmasks the tumor suppressor roles of these EGFR-regulated miRNAs. Mol Cancer Res; 9(7); 960–75. ©2011 AACR.