Barrett esophagus (BE) is a human metaplastic condition that is the only known precursor to esophageal adenocarcinoma. BE is characterized by a posterior intestinal-like phenotype in an anterior organ and therefore it is reminiscent of homeotic transformations, which can occur in transgenic animal models during embryonic development as a consequence of mutations in HOX genes. In humans, acquired deregulation of HOX genes during adulthood has been linked to carcinogenesis; however, little is known about their role in the pathogenesis of premalignant conditions. We hypothesized that HOX genes may be implicated in the development of BE. We demonstrated that three midcluster HOXB genes (HOXB5, HOXB6, and HOXB7) are overexpressed in BE, compared with the anatomically adjacent normal esophagus and gastric cardia. The midcluster HOXB gene signature in BE is identical to that seen in normal colonic epithelium. Ectopic expression of these three genes in normal squamous esophageal cells in vitro induces markers of intestinal differentiation, such as KRT20, MUC2, and VILLIN. In BE-associated adenocarcinoma, the activation midcluster HOXB gene is associated with loss of H3K27me3 and gain of AcH3, compared with normal esophagus. These changes in histone posttranslational modifications correlate with specific chromatin decompaction at the HOXB locus. We suggest that epigenetically regulated alterations of HOX gene expression can trigger changes in the transcriptional program of adult esophageal cells, with implications for the early stages of carcinogenesis.