Purpose: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has attracted extensive attention in various types of malignant tumors. However, the role of DNA-PKcs in cutaneous squamous cell carcinoma (cSCC) development has not been elucidated. In this study, we investigated the role of DNA-PKcs in cSCC and the molecular mechanisms of TGF-beta 1-induced cSCC progression mediated by DNA-PKcs. Methods: We performed bioinformatic analysis and RT-PCR to examine the DNA-PKcs expression level in cSCC. Then, we downregulated DNA-PKcs using a DNA-PK-specific inhibitor or small interfering RNA (siRNA) to explore the effects of DNA-PKcs on SCL-1 cell migration and invasion. To further investigate the mechanism by which DNA-PKcs promotes cSCC progression, TGF-beta 1 and the TGF-beta receptor (TGF-beta R) I/II dual inhibitor LY2109761 were used to examine whether DNA-PKcs participates in TGF-beta 1/Smad signaling. Results: DNA-PKcs expression was upregulated in cSCC. DNA-PK inhibition or expression knockdown resulted in inhibited migration and invasion and altered epithelial-mesenchymal transition (EMT) marker expression patterns in SCL-1 cells. Importantly, TGF-beta 1 mediated EMT induction in cSCC cells, and DNA-PKcs was identified as a TGF-beta 1-responsive gene. TGF-beta 1 promoted DNA-PKcs transcription, and DNA-PKcs enhanced the TGF-beta 1-induced EMT program involved in cSCC invasion and metastasis by phosphorylating Smad3. Conclusion: This study is the first to show that DNA-PKcs mediates EMT to promote cSCC aggressiveness by targeting the TGF-beta 1/Smad signaling pathway, which provides insight into how DNA-PKcs impacts cSCC progression and identifies a new therapeutic target.