Squamous cell carcinoma (SCC) is defined as a category of aggressive malignancies arising from the squamous epithelium of various organs. Resistance to chemotherapies is a common feature of SCCs, which leads to a poor prognosis among SCC patients. Recently, studies have illustrated the essential tumor suppressive role of ARID1A in several cancer types, but its role in SCCs remains unclear. Cancer stemness has been recognized as a main reason for tumorigenesis and is commonly correlated with chemoresistance, yet the relationship between ARID1A and cancer stemness remains unknown. In this study, we showed that Arid1a conditional knockout mice had a high incidence of SCCs occurring in the tongue and esophagus. ARID1A depletion promoted tumor initiation and cancer stemness in human SCC cells. Mechanistic studies revealed that ARID1A blocked the interaction between cyclin-dependent kinases (CDKs) and retinoblastoma protein (Rb), reducing the phosphorylation of Rb. Dephosphorylated Rb suppressed E2F1 activity and then suppressed cancer stemness by inactivating c-Myc. Furthermore, we showed that ARID1A depletion significantly increased the chemoresistance of SCC and that a CDK inhibitor exhibited a favorable effect on rescuing the chemoresistance caused by ARID1A loss. Collectively, our study showed that ARID1A inhibits the cancer stemness of SCCs by competing with CDKs to bind with Rb to inhibit the E2F1/c-Myc pathway.