Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, the precise mechanism of T. gondii restriction is not known. Using automated spatially targeted optical micro proteomics we determined that inducible nitric oxide synthetase (iNOS) was highly enriched at GBP-positive PV in murine macrophages. iNOS expression in myeloid cells was necessary to control T. gondii growth in vivo and in vitro. iNOS activity was dispensable for GBP2 recruitment and PV membrane ruffling, however, these vacuoles contained dividing parasites. T. gondii restriction by iNOS required nitric oxide, nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with the chromosome 3 GBPs to target distinct biology of the PV that are necessary for optimal parasite clearance in murine myeloid cells.