The present study conducted an immunohistochemical investigation of cyclin D1 and Ki-67 expression in oral squamous cell carcinoma (SCC) to evaluate the correlations between cell differentiation, cell proliferation and metastasis, and the effect of anticancer drug medication and cyclin D1 expression. Cyclin D1 and Ki-67 were detected clearly in the nuclei of 35 SCC samples. No correlation between cyclin D1 protein expression and oral SCC differentiation was found. By contrast, the majority of metastatic foci (90%) exhibited strong cyclin D1 expression, whereas weak expression was observed in metastatic foci with pre-operative adjuvant therapy. Additionally, cyclin D1 and Ki-67 were expressed in basal to suprabasal cells of well-differentiated oral SCC, whereas cyclin D1-positive and Ki-67-negative cells were present in the highly-differentiated region, according to a double-immunostaining method. These results indicate that the expression of cyclin D1 protein plays a role in cell differentiation and cell proliferation in well-differentiated oral SCC.