Chromosome 1 open reading frame 10 (C1orf10) is a recently identified gene encoding a protein with an S100 EF-hand calcium-binding motif, and its expression is known to be down-regulated in esophageal squamous cell carcinoma. In this study, to determine whether the loss of C1orf10 gene function could contribute to the development of oral squamous cell carcinoma (OSCC), we have evaluated the expression status of this gene by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative real-time PCR analysis. A high frequency of decrease in C1orf10 gene was detected not only in OSCC-derived cell lines but also in tumor tissues. Next, to define biological function of this gene in oral carcinogenesis, we transfected Clorf10 with an Ecdysone-inducible system in OSCC cell lines and analyzed the effects of its overexpression. Induction of C1orf10 expression resulted in a significant decline in the rate of cell proliferation, and in an arrest in the G1 phase of the cell cycle, with a down-regulation of Cyclin D1 expression. However, we could not detect significant difference in the percentage of apoptotic cells. Thus, our results suggest that the down-regulation of C1orf10 gene plays a role in oral carcinogenesis, and that its expression may negatively regulate OSCC cell proliferation by arresting the cell cycle.