Cancer stem cells (CSCs) are a small subpopulation of the overall cancer cell population and display enhanced tumorigenicity, metastasis, and drug resistance, which make them plausible targets for cancer therapies. Dual-specificity tyrosine kinases (DYRK1-4) are a family of protein kinases involved in cancer development; however, their role in oral cancer and stemness remains unknown. In this study, we investigated the role of DYRK in oral carcinogenesis by screening the expression of the DYRK isoforms in normal human oral keratinocytes and oral squamous cell carcinomas (OSCC). Among DYRK members, DYRK1A was the dominant DYRK isoform in oral epithelial cells and was upregulated in OSCC. Genetic deletion and chemical inhibition of DYRK1A suppressed aggressive tumor phenotypes, such as tumor growth in vitro and in vivo, migration, and chemoresistance. DYRK1A was also essential for self-renewal of CSCs and was highly expressed in CSC-enriched populations. The DYRK1A deletion inhibits FGF2 production in OSCC and concomitantly suppresses ERK signaling pathway, a downstream signaling activated by FGF2. Furthermore, FGF2 treatment in DYRK1A-deleted OSCC cells rescued CSC phenotype. Our study first identifies DYRK1A as a novel molecular determinant for oral cancer stemness and aggressiveness through FGF2/ERK signaling. Since DYRK1A is chemically modifiable by harmine, our study suggests that DYRK1A may be a potential therapeutic target for eradicating oral CSCs.