Control of tumor growth by adaptive immune cells requires the recognition of tumor antigens. Beside an altered proteome that provides peptide neo-antigens, tumors show an altered lipidome, potentially supplying novel or overexpressed lipid antigens that are recognized by natural killer T cells (NKT cells). NKT cells are a heterogeneous population of immunoregulatory lymphocytes that recognize foreign as well as self-lipids that are presented on the surface of antigen-presenting cells (APC) via CD1d molecules. Whereas type I NKT cells mainly induce protective immunity, type II NKT cells suppress anti-umor immune responses. We therefore hypothesized the presence of type II NKT antigens in tumors that limit anti-tumor immunity. To identify such tumor-enriched lipid antigens we performed a comparative mass spectrometric screening of CD1d-binding lipids from autochthonous mouse mammary tumors and normal mammary glands. Of 31 CD1d-bound lipids that were enriched in mammary tumors compared to normal tissue a surprisingly high proportion were ether lipids, most of them exhibiting a serine headgroup. These ether-linked phosphatidylserine (ePS) species showed the highest overabundance in murine mammary carcinomas. Accordingly, PS synthesis pathways are overexpressed in breast cancer. Moreover, enhanced expression of phosphatidylserine synthase 1 (PTDSS1) correlates with a poor prognosis for breast cancer patients. By using ether-PS-loaded CD1d tetramers, a new subset of type II NKT cells recognizing the serine headgroup was identified. Such NKT cells recognizing ePS showed an intrinsically reduced inflammatory potential. Analysis of PTDSS1-knockdown tumors grown in syngeneic mice confirmed a role for PS in anti-tumor immunity. In conclusion, we provide evidence for the existence of tumor-enriched lipid antigens that impact tumor development by affecting anti-tumor immunity.