IL-27 and TCRγδ+ T lymphocytes play critical roles in both innate and adaptive immune responses in health and disease, including infection and tumors. Although the activity of IL-27 is well characterized in different human immune cells, no information is available on the role of IL-27 in human TCRγδ+ T lymphocytes. Here, we provide the first evidence that TCRγδ+ T lymphocytes express both gp130 and WSX-1 chains of IL-27R, and that IL-27 may function in TCRγδ+ T cells by (i) inducing STAT1 and STAT3 phosphorylation, (ii) stimulating cytotoxicity against tumor cells through upregulation of cytotoxic granules production, (iii) reducing the release of Th2-related cytokines, such as IL-5 and IL-13, and inducing IFN-γ production, and (iv) upregulating the expression of CD62L. These results highlighted a novel immunoregulatory property of human IL-27 that may be relevant in the immune response against tumors. Our results may offer new perspectives for the development of future clinical trials using IL-27 and TCRγδ+ cells for cancer immunotherapy.