Background: Cutaneous squamous cell carcinoma (SCC) is an easily occurred cancer, which can worsen the quality of life considerably. It is known that external stimulus induces cutaneous SCC via provoking oxidative stress. NAD(P)H dehydrogenase 1 (NQO1) has functions as a guardian against oxidative stress. However, the effect of NQO1 on cutaneous SCC is not clearly elucidated. Objectives: In this study, we investigated the effect of NQO1 on cutaneous SCC cells. Methods: To examine the effects of NQO1, we transduced SCC lines (SCC12 and SCC13) with the NQO1 expressing or knockdown adenovirus. Results: Overexpression of NQO1 resulted in significant decrease of cell proliferation and colony forming activity of SCC lines. By contrast, knockdown of NQO1 increased the cell proliferation and colony forming activity. Accordingly, the levels of proliferation-related regulators, such as CDK4, CDK6, SOX2 and p63, were decreased by overexpression of NQO1, while those were increased by knockdown of NQO1. In addition, NQO1 affected the invasion and migration of SCC cells in a very similar way, with the regulation of epithelial-mesenchymal transition (EMT)-related molecules, including E-cadherin, N-cadherin, Vimentin, Snail and Slug. Finally, overexpression of NQO1 decreased the level of phosphorylated AKT, JNK and p38 MAPK, while knockdown of NQO1 increased the level of phosphorylated signaling molecules. Conclusion: Based on these data, NQO1 has tumor suppressive function in cutaneous SCC cells.