This study was aimed to investigate the antiproliferative effect of escopoletin on CAL 27 and CAL 33 oral squamous cancer cell lines. MTT assay and flow cytometry were used for the analysis of escopoletin effect on cell viability and apoptosis, respectively. Western blot analysis was used for the examination of cyclin and cyclin-dependent kinase expression after treatment of CAL 33 cells with escopoletin. The results showed a marked decrease in the viability of CAL 27 and CAL 33 cell lines after 48 h of escopoletin treatment. Treatment of CAL 33 cells with escopoletin led to the induction of apoptosis and arrest of cell cycle at G0/G1 phase. In the cells treated with escopoletin cyclin D1 and E expression was reduced and CDK1 expression was inactivated. The above findings suggest that escopoletin exhibits inhibitory effect on the oral squamous cancer through induction of apoptosis and arrest of cell cycle. Therefore escopoletin can be a promising candidate for the prevention of oral squamous cancer.