Krüppel-like factor 5 (Klf5; also called IKLF or BTEB2), a zinc-finger transcription factor with proproliferative and transforming properties in vitro, is expressed in proliferating cells of gastrointestinal tract epithelia, including in basal cells of the esophagus. Thus, Klf5 is an excellent candidate to regulate esophageal epithelial proliferation in vivo. Nonetheless, the function of Klf5 in esophageal epithelial homeostasis and tumorigenesis in vivo has not previously been determined. Here, we used the ED-L2 promoter of the Epstein-Barr virus to express Klf5 throughout esophageal epithelia. ED-L2/Klf5 transgenic mice were born at the appropriate Mendelian ratio, survived to at least 1 yr of age, and showed no evidence of esophageal dysplasia or cancer. Staining for bromodeoxyuridine (BrdU) demonstrated increased proliferation in the basal layer of ED-L2/Klf5 mice, but no proliferation was seen in suprabasal cells, despite ectopic expression of Klf5 in these cells. Notably, expression of the KLF family member Klf4, which binds the same DNA sequences as Klf5 and which inhibits proliferation and promotes differentiation, was not altered in ED-L2/Klf5 transgenic mice. In primary esophageal keratinocytes that overexpressed Klf5, expression of Klf4 still inhibited proliferation and promoted differentiation, providing a possible mechanism for the persistence of keratinocyte differentiation in ED-L2/Klf5 mice. To identify additional targets for Klf5 in esophageal epithelia, we performed functional genomic analyses and identified a total of 15 differentially expressed genes. In summary, while Klf5 positively regulates proliferation in basal cells, it is not sufficient to maintain proliferation in the esophageal epithelium.