Abstract Natural killer (NK) cells safeguard against early tumor formation by seeking out and destroying transformed target cells in a process referred to as NK immunosurveillance. While it is clear that malignant brain tumors such as glioblastoma (GBM) evade NK-mediated tumor suppression, the precise mechanisms by which this occurs remain unknown. We now show that shRNA-mediated knockdown of the β-galactoside-binding lectin, galectin-1 (gal-1), in malignant glioma cells leads to the failure to form lethal intracranial tumors in RAG1-/- mice, a mouse strain devoid of adaptive immunity. However, gal-1 deficient glioma growth is fully restored on implantation into the brain of severely immunocompromised NOD-scid IL2Rg null mice, which lack both adaptive and innate immune function, thus implicating the innate immune response in the early rejection of gal-1 deficient glioma. Immunodepletion of NK cells in RAG1-/- or C57BL/6J mice using anti-asialo GM1 or anti-NK1.1 antibodies permit the growth of large gal-1 deficient gliomas, while macrophage depletion with clodronate liposomes only permits limited tumor growth. This combined result suggests that NK cells and macrophages may work together to achieve gal-1 deficient glioma rejection. Antigen-specific IFN-γ ELISpot assays using splenocytes from immunocompetent C57BL/6J mice indicate that gal-1 deficient glioma is cleared prior to the onset of an adaptive anti-tumor immune response. Flow cytometric analysis of brain tumor-infiltrating immune cells reveal that gal-1 deficient gliomas contain significantly more macrophages and granzyme B+ NK cells compared to gal-1 expressing gliomas. In-vitro experiments further show that gal-1 deficient glioma cells are inherently over 3-times more sensitive to NK-mediated tumor lysis, fail to suppress pro-inflammatory (M1) microglial activation, and secrete pro-inflammatory cytokines IL-1β, IL-12p70, and CXCL2. We conclude that glioma-derived gal-1 is a powerful inhibitor of NK-mediated cytotoxicity in-vivo, and predict that its suppression will be of therapeutic value in the treatment of human malignant brain tumors by dramatically heightening anti-tumor NK immunosurveillance. Citation Format: Gregory J. Baker, Viveka Nand Yadav, Peter Chockley, Robert Doherty, Michael Ritt, Sivaraj Sivaramakrishnan, Maria G. Castro, Pedro R. Lowenstein. Natural killer cells eradicate galectin-1 deficient glioma in the absence of adaptive immunity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3651. doi:10.1158/1538-7445.AM2014-3651