Histone deacetylase (HDAC) activity plays the role of deacetylation of histone and non-histone proteins, which can alter gene expression patterns and cell behavior potentially associated with malignant transformation. Aberrant expression of HDAC1 has been found in various types of cancers, which indicated that it might be a target for cancer therapy. In this study, overexpression of HDAC1 was found in esophageal cancer samples by real-time RT-PCR, compared with adjacent non-cancerous tissues. To further verify the possibility of anticancer treatment by silencing the increased HDAC1 in esophageal carcinoma cells, HDAC1 expression was knockdown using plasmid-based RNA interference (RNAi). Results showed the HDAC1 expression was efficiently inhibited and the acetylation of histone H3 was significantly increased by RNAi in EC109 cells. Increased apoptotic cell death was observed when HDAC1 expression was knockdown, which indicated that cells were more sensitive to radiation. Moreover, the results also showed DNA was more easily broken by radiation in EC109 cells when HDAC1 expression was knockdown, as measured by γH2AX foci and single-cell electrophoresis. Our data suggested that targeting the increased HDAC1 expression might be feasible for esophageal cancer therapy.