Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for similar to 20% of annual skin cancer-associated mortalities. Accumulating evidence demonstrates that the dysregulation of micro (mi) RNAs serves a significant role in the tumorigenesis and progression of human cSCC. MicroRNA-31 (miR-31) is upregulated in cSCC and is involved in cSCC development. However, the underlying mechanism remains unclear. The present study demonstrated that miR-31 is upregulated in the cSCC cell line, A-431, and that miR-31 expression contributes to the cell proliferation and invasion of cSCC. In addition, bioinformatics combined with dual luciferase reporter analysis was applied to determine that the tumor suppressor RhoTBT1 was a direct target of miR-31. In addition, miR-31 mimics reduced RhoBTB1 expression in A-431 cells. The results of MTT and Transwell assays demonstrated that knockdown of RhoBTB1 by short interfering RNA induced cell proliferation and invasion in A-431 cells. These results indicated that suppression of RhoBTB1 may be involved in cSCC tumorigenesis, which was directly affected by miR-31. In conclusion, the present study provides evidence that miR-31 acts as an oncogene through direct repression of RhoTBT1 expression in cSCC cancer, suggesting a potential application of miR-31 in prognosis prediction and its therapeutic application in cSCC.