IL-21 is a cytokine that can promote the anti-tumor responses of the innate and adaptive immune system. Mice treated with IL-21 reject tumor cells more efficiently, and a higher percentage of mice remain tumor-free compared with untreated controls. In this study, we demonstrate that in certain tumor models IL-21-enhanced tumor rejection is NKG2D dependent. When engagement of the NKG2D receptor was prevented, either due to the lack of ligand expression on the tumor cells or due to direct blocking with anti-NKG2D mAb treatment, the protective effects of IL-21 treatment were abrogated or substantially diminished. Specifically, IL-21 only demonstrated a therapeutic effect in mice challenged with a retinoic acid early inducible-1δ-bearing lymphoma but not in mice bearing parental RMA tumors lacking NKG2D ligands. Furthermore, treatment with a blocking anti-NKG2D mAb largely prevented the therapeutic effect of IL-21 in mice challenged with the 4T1 breast carcinoma, the 3LL lung carcinoma, and RM-1 prostate carcinoma. By contrast, IL-21 did mediate beneficial effects against both the parental DA3 mammary carcinoma and DA3 tumors transfected with H60, a NKG2D ligand. We also observed that IL-21 treatment could enhance RMA-retinoic acid early inducible-1δ tumor rejection in RAG-1−/− deficient mice, thereby demonstrating that the IL-21-induced protective effect can be mediated by the innate immune system and that, in this case, IL-21 does not require the adaptive immune response. Collectively, these findings suggest that IL-21 therapy may work optimally against tumors that can elicit a NKG2D-mediated immune response.