Epithelial cell adhesion molecule (EpCAM), involved in Ca2+-independent homotypic cell-cell adhesion in epithelial tissues, is overexpressed in several cancer types. Although studies investigating the function of EpCAM in cancers have shown that it plays a role in cell proliferation, invasion and metastasis, the overall function of EpCAM in cancer cells has remained elusive. Here, we report a novel function of EpCAM in multicellular aggregates (MCAs). EpCAM inhibition using RNA interference (RNAi) did not affect cell morphology, proliferation or expression of certain genes, including cyclin D1 in monolayer cultures of the human oral squamous cell carcinoma cell lines HSC-3 or HSC-4. However, in HSC-4 cells cultured as MCAs, suppression of EpCAM significantly reduced the expression levels of cyclin D1. Nuclear localization of the cyclin D1 protein was observed in MCAs of HSC-4 cells but not in MCAs of EpCAM knockdown HSC4 cells, suggesting that EpCAM regulates cyclin D1 expression and localization in HSC-4 cells under anchorage-independent conditions. We propose that targeting EpCAM might result in more efficient therapies under certain conditions of oral squamous cell carcinoma.