C-terminal binding protein-2 (CtBP2), as a transcriptional co-repressor, has been shown to mediate the repression of p16INK4A, a tumor suppressor gene product, in primary human cells. Here we aimed to investigate how the correlation between CtBP2 and p16INK4A influenced the development of esophageal squamous cell carcinoma (ESCC). Immunohistochemistry of ESCC tissue sections indicated that the CtBP2 and p16INK4A expressions were inversely correlated to each other with a linear regression coefficient of −0.747 (P < 0.05), and Western blot analysis revealed that CtBP2 was higher expressed in tumorous tissues than in adjacent non-tumorous tissues. Either CtBP2 or p16INK4A expression was significantly related to histological differentiation (P = 0.016 or 0.001) and to the expression of Ki-67, a proliferating marker (P = 0.006 or 0.02), and patients with higher CtBP2 and lower p16INK4A expressions had shorter overall survival. We also observed that CtBP2 modulated the cell proliferation and cell cycle in ECA109 cells, an ESCC cell line, by inhibiting p16INK4A. Overexpression or knockdown of CtBP2 in ECA109 cells was found to inhibit or activate the mRNA or protein expression of p16INK4A, which in turn altered the cell proliferation and cell cycle in ECA109 cells, as measured by flow cytometry and cell count assay. Additionally, after ECA109 cells silenced for CtBP2 were treated with cisplatin (an anti-ESCC agent), the p16INK4A expression was up-regulated, and the cell apoptosis was promoted, thus confirming the repression of p16INK4A by CtBP2. Collectively, all results suggested that CtBP2 might contribute to the progression of ESCC through a negative transcriptional regulation of p16INK4A. J. Cell. Biochem. 114: 1343–1354, 2013. © 2013 Wiley Periodicals, Inc.