KLF5 is an essential basic transcriptional factor that regulates a number of physiopathological processes. In this study, we tested whether and how KLF5 modulates the epithelial-mesenchymal transition (EMT). Using transforming growth factor β (TGF-β)- and epidermal growth factor (EGF)-treated epithelial cells as an established model of EMT, we found that KLF5 was downregulated during EMT and that knockdown of KLF5 induced EMT even in the absence of TGF-β and EGF treatment, as indicated by phenotypic and molecular EMT properties. Array-based screening suggested and biochemical analyses confirmed that the microRNA 200 (miR-200) microRNAs, a group of well-established EMT repressors, were transcriptionally activated by KLF5 via its direct binding to the GC boxes in miR-200 gene promoters. Functionally, overexpression of miR-200 prevented the EMT induced by KLF5 knockdown or by TGF-β and EGF treatment, and ectopic expression of KLF5 attenuated TGF-β- and EGF-induced EMT by rescuing the expression of miR-200. In mouse prostates, knockout of Klf5 downregulated the miR-200 family and induced molecular changes indicative of EMT. These findings indicate that KLF5 maintains epithelial characteristics and prevents EMT by transcriptionally activating the miR-200 family in epithelial cells.