Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.