It has been shown previously that the suppression of tumor immunosurveillance may be a mechanism by which tumors resist immune detection and elimination. In this study, we evaluated the role of the immunoregulatory natural killer T (NKT) cells in the biology of immunosurveillance of osteosarcoma. The K7M2 mouse osteosarcoma cell line was implanted orthotopically into wild-type and NKT cell–deficient CD1d knockout (KO) BALB/c mice, and mice were monitored for growth of primary tumors. Further, we examined the role of CD4⁺ and/or CD8⁺ cells by depleting the cells in vivo and measuring CTL activity in vitro. We also asked the role of interleukin (IL)-4 receptor α (IL-4Rα)-signal transducer and activator of transcription 6 (STAT6) signaling, including IL-13, and transforming growth factor β (TGF-β) by using gene-disrupted mice or treating mice with cytokine antagonists. We were surprised to find a high rate of rejection of osteosarcoma primary tumors in 88% (14 of 16) of CD1d KO mice compared with syngeneic wild-type BALB/c mice that showed rejection of tumor in <24% of mice. Further studies suggested that the rejection of tumor in CD1d KO mice was dependent on CD8⁺ lymphocytes. Distinct from other murine tumor models, the negative regulation induced by CD1d-restricted NKT cells was not dependent on IL-4Rα-STAT6 signaling, including IL-13, or on TGF-β. These data suggest that a novel CD1d-restricted NKT cell–mediated mechanism for tumor immunosuppression is active in the K7M2 osteosarcoma model and that NKT cells can regulate immunosurveillance through more than one pathway. (Cancer Res 2006; 66(7): 3869-75)