Gut-enriched Kruppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract, and its expression is associated with growth arrest. Previous work showed that GKLF expression was down-regulated in esophageal squamous cancer. In the present work, the effects of sense and antisense expression of GKLF on the esophageal squamous cancer cell line EC9706 were observed to get more knowledge of the role of down-regulation of GKLF in the initiation and/or progression of esophageal cancer. Antisense transfection of GKLF gene in EC9706 led to decreased expression of GKLF in the cells, and these cells showed increased cell growth rate, indicating that down-regulation of the GKLF gene might contribute to cellular hyperproliferation. Both increased and decreased expression of GKLF in EC9706 by sense and antisense GKLF expression also led to the decreased adhesion of cells, suggesting a role of GKLF in the metastasis of esophageal squamous cancer. The results demonstrate that down-regulation of GKLF expression in esophageal squamous cancer causes the cancer cells to become hyperproliferative and less adhesive, and suggest that decreased expression of GKLF may contribute to the malignant phenotype of esophageal squamous cancer.