Epithelial adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in intercellular adhesion. In particular, EpCAM appears to be overexpressed by the majority of human epithelial carcinomas, including colorectal, breast, head and neck, and hepatic carcinomas. We therefore hypothesized that EpCAM would be a good molecular target for cancer gene therapy. EpCAM protein expression in 48 primary tongue cancers and 10 normal oral mucosa was evaluated using anti-EpCAM immunohistochemistry, and correlation was examined with the clinicopathologic factors. In four human tongue cancer cell lines (SAS, HSC-2, OSC19 and OSC20), we investigated EpCAM expression by reverse transcription-polymerase chain reaction (RT-PCR). The invasive potential of cancer cells was evaluated using Matrigel invasion assay. Moreover, the effect of EpCAM inhibition was analyzed using RNA interference (RNAi). EpCAM overexpression was detected in 30 of 48 tongue cancers (62.5%), and was significantly higher in primary squamous cell carcinoma (SCC) of the tongue than in normal oral mucosa. The expression of EpCAM was significantly associated with tumor size, regional lymph node metastasis, histological differentiation and invasion pattern. Cancer cell lines with higher EpCAM expression had more invasive potential. Moreover, RNAi-mediated EpCAM reduction decreased the invasion potential and proliferation activity. These results indicated that the overexpression of EpCAM was correlated with a more aggressive phenotype of tongue cancer. Moreover, we suggested that EpCAM could be a molecular target, and that RNAi targeting EpCAM could be useful for tongue cancer gene therapy.