Epithelial cell adhesion molecule (EpCAM) is important for cell proliferation and differentiation but mechanisms regulating their expression are unclear. Because EpCAM may play a role in carcinogenesis, we investigated the clinicopathologic significance of its expression in oral squamous cell carcinoma (OSCC) and the involvement of DNA methylation machinery in regulation of EpCAM expression during tumorigenesis. Immunohistochemical staining for EpCAM expression and DNA methyltransferase-1 (DNMT1) was done in 112 OSCC cases. Tumor genomic DNA was extracted and EpCAM promoter methylation was examined by methylation-specific polymerase chain reaction in 72 OSCC specimens. Immunoreactivity and methylation were correlated with clinicopathologic features. EpCAM expression was undetectable in normal epithelium; high expression was observed in 51% (57/112) of OSCC. Heterogeneity and plasticity of EpCAM expression was observed during tumor development. Allele methylation was found in 51% (37/72) of OSCC cases analyzed. EpCAM expression was associated with promoter methylation (p = 0.008). However, EpCAM expression and promoter methylation did not correlate with clinicopathologic OSCC variables. DNMT1 expression was occasionally observed in basal cells of normal epithelium; high expression was observed in 47% (53/112) of OSCC. DNMT1 did not correlate with EpCAM expression or methylation status. High DNMT1 expression correlated with tumor size (p < 0.0001) histologic differentiation (p = 0.012) and clinical stage (p < 0.0001) of OSCC. EpCAM expression increased during development of OSCC. EpCAM promoter methylation is associated with EpCAM expression levels in OSCC, suggesting an epigenetically mediated regulation of EpCAM expression. Increased DNMT1 protein expression may be involved in histogenesis and progression of OSCC.