Glutamine is a conditionally essential amino acid consumed by rapidly proliferating cancer cells, which deprives the same fuel from immune cells and contributes to tumor immune evasion. As such, the broad antagonism of glutamine in tumors and the tumor microenvironment may lead to direct antitumor activity and stimulation of antitumoral immune responses. DRP-104 (sirpiglenastat) was designed as a novel prodrug of the broad-acting glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). DRP-104 is an inactive form that is preferentially converted to DON within tumors. Metabolomic profiling of tumors treated with DRP-104 revealed widespread changes indicative of the disruption of tumor anabolism and canonical cancer metabolism pathways; including altered glutamine metabolism while several immunosuppressive metabolites were decreased. Gene expression profiling revealed broad immunological modulation, confirmed by flow cytometry indicating that DRP-104 treatment resulted in substantial and broad changes in various immune cell infiltrates, such as increased TIL, T, NK, and NK T cells. Functionally, T cells became more proliferative and less exhausted; tumor-associated macrophages were polarized to the M1 phenotype; MDSCs and protumorigenic proteins were decreased in TME. Finally, DRP-104 demonstrated significant antitumor activity as a monotherapy, which was further enhanced in combination with checkpoint blockade therapies, leading to improved survival and long-term durable cures. In summary, DRP-104 broadly remodels the tumor microenvironment by inducing extensive tumor metabolism effects and enhancing the infiltration and function of multiple immune cells distinct from those obtained by checkpoint inhibitor therapy. This unique mechanism of action supports the ongoing clinical development of DRP-104 alone and in combination with checkpoint inhibitors.