Apurinic/apyrimidinic endonuclease-1 (APE-1), a key enzyme responsible for DNA base excision repair (BER), has been linked to cancer chemoradiosensitivity. The phosphorylation of p65 plays a role in the activation of this pathway. In this study, we investigated APE-1 expression and its interaction with p65 in esophageal squamous cell carcinoma (ESCC) tissue. The expression of APE-1, p65, p65 nuclear localization sequence (p65-NLS), and monocyte chemoattractant protein-1 (MCP-1) was assessed by immunohistochemical analysis in 67 human ESCC tissue samples. Real-time PCR and western blotting were also performed. p65 siRNA was evaluated to determine the role of p65 in the regulation of APE-1 expression. We found nuclear localization of APE-1 in 89.6% (60/67) of ESCC tissue samples. We also observed the colocalization of p65-NLS and APE-1 in esophageal cancer tissue. In KYSE220 cells, pretreatment of MG-132 significantly abrogated upregulation of p65 and APE-1 levels induced by MCP-1, and treatment with 10 and 20 nM p65 siRNA significantly inhibited APE-1 mRNA expression. siRNA for p65 treatment significantly increased the apoptotic index in 5-FU-treated KYSE220 cells. We conclude that APE-1 is overexpressed and mainly localized in the nuclear compartment of cancer cells, and partly regulated by p65 in the NF-κB pathway in ESCC tissue.