Abstract The presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) poses a serious obstacle to cancer treatment. The bioactive lysophospholipid sphingosine 1-phosphate (S1P), which is produced by sphingosine kinases (SphK1 or SphK2), regulates tumor and T cell survival and activity. Thus, it has been targeted to improve the outcome of anti-cancer therapeutics. However, S1P has not yet been investigated in terms of how it regulates myeloid cell activity. The present study demonstrates that deletion of SphK2-mediated S1P in MDSCs favorably modifies the tumor microenvironment and renders infiltrating myeloid cells with antigen presenting phenotype, leading to increased ability to control tumor growth. Furthermore, in preclinical prostate and melanoma cancer models, the combination of PD1 antibody and SphK2 pharmacological inhibitor (ABC294640) dramatically reduced tumor growth and increased the survival of tumor bearing mice. Mechanistically, the S1P pulldown and subsequent MS-based proteomics analysis revealed that S1P binds to various proteins involved in metabolic and ribosomal biogenesis pathways. Notably, both the isoforms of Acetyl CoA Carboxylase, the rate-limiting fatty acid metabolic enzyme, binds to S1P. Furthermore, investigations using deletion, overexpression, and site-directed mutagenesis demonstrated that ACC1 activity is necessary to preserve the immunogenic capacity of SphK2 KO MDSCs. This study highlights the clinical value of manipulating MDSC suppressive activity by targeting the S1P/ACC1 axis to boost T cell-mediated tumor control and highlights the crucial function that S1P plays as an immunoregulatory mediator in MDSCs. Supported grants from NIH (R01 CA250458, R01 CA236379)