The cutaneous squamous cell carcinoma (cSCC) originates from epithelial stem cells through the dysregulation of self-renewal and differentiation. Recent studies have identified methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m(6)A) modification as key regulator of fate of stem cells. However, little is known about the functional importance of METTL3 in cSCC. Here, Western blot and immunohistochemistry were used to investigate the METTL3 levels in cSCC tissues. Functional experiments including surface marker detection, Brdu incorporation assay, colony forming assay, m(6)A dot blot and tumor xenograft assay were performed to investigate the properties in cSCC cell lines after METTL3 knock down. The expression of METTL3 was up-regulated in cSCC samples. METTL3 knock down impaired cSCC cell stem-like properties, including colony forming ability in vitro and tumorigenicity in vivo. Furthermore, METTL3 knock down and methylation inhibitor cycloleucine could decrease the m(6)A levels and the expression of Delta Np63 in cSCC. Exogenous expression of Delta Np63 partially restored the cell proliferation of METTL3-knockdown cSCC cells. Therefore, our data indicated the m(6)A methyltransferases METTL3 as a critical gene in regulating tumorigeneis of cSCC. (C) 2019 Elsevier Inc. All rights reserved.