Natural killer (NK) cells and T cells are key effectors of antitumor immune responses and major targets of checkpoint inhibitors. In multiple cancer types, we find that the expression of Wnt signaling potentiator R-spondin genes (e.g., RSPO3) is associated with favorable prognosis and positively correlates with gene signatures of both NK cells and T cells. Although endothelial cells and cancer-associated fibroblasts comprise the R-spondin 3-producing cells, NK cells and T cells correspondingly express the R-spondin 3 receptor LGR6 within the tumor microenvironment (TME). Exogenous expression or intratumor injection of R-spondin 3 in tumors enhanced the infiltration and function of cytotoxic effector cells, which led to tumor regression. NK cells and CD8+T cells independently and cooperatively contributed to R-spondin 3-induced control of distinct tumor types. The effect of R-spondin 3 was mediated in part through upregulation of MYC and ribosomal biogenesis. Importantly, R-spondin 3 expression enhanced tumor sensitivity to anti-PD-1 therapy, thereby highlighting new therapeutic avenues.SIGNIFICANCE: Our study identifies novel targets in enhancing antitumor immunity and sensitizing immune checkpoint inhibition, which provides a rationale for developing new immunotherapies against cancers. It also offers mechanistic insights on Wnt signaling-mediated modulation of anticancer immu-nity in the TME and implications for a putative R-spondin-LGR6 axis in regulating NK-cell biology.