Neurotransmitter receptors are increasingly recognized to play important roles in anti-tumor immunity. The expression of the ion channel N-methyl-D-aspartate receptor (NMDAR) on macrophages was reported, but the role of NMDAR on macrophages in the tumor microenvironment (TME) remains unknown. Here, we show that the activation of NMDAR triggered calcium influx and ROS production, which fueled immunosuppressive activities in tumor-associated macrophages (TAMs). NMDAR channel blockers, MK-801, memantine and magnesium, effectively suppressed these processes in TAMs. mRNA sequencing and Single-cell RNA-sequencing analysis revealed that blocking NMDAR functionally and metabolically altered TAM phenotypes, such that they could better promote T cell- and NK cell-mediated anti-tumor immunity. Treatment with NMDAR channel blockers in combination with anti-PD-1 antibody led to elimination of the majority of established preclinical tumors. Thus, our study uncovered an unknown role for NMDAR in regulating macrophages in the TME and provide a rational for targeting NMDAR for tumor immunotherapy.