During an immune response, macrophages specifically reprogramme their metabolism to support functional changes. Here, we revealed that nucleotide metabolism is one of the most significantly reprogrammed pathways upon classical activation. Specifically, de novo synthesis of pyrimidines is maintained up to uridine monophosphate, but blocked at cytidine triphosphate and deoxythymidine monophosphate synthesis; de novo synthesis of purines is shut off at the last step (catalysed by AICAR transformylase/IMP cyclohydrolase, ATIC), and cells switch to increased purine salvage. Nucleotide degradation to nitrogenous bases is upregulated but complete oxidation of purine bases (catalysed by xanthine oxidoreductase, XOR) is inhibited, diverting flux into salvage. Mechanistically, nitric oxide was identified as a major regulator of nucleotide metabolism, simultaneously driving multiple key changes, including the transcriptional downregulation of Tyms and profound inhibition of ATIC and XOR. Inhibiting purine salvage using Hgprt knockout or inhibition alters the expression of many stimulation-induced genes, suppresses macrophage migration and phagocytosis, and increases the proliferation of the intracellular parasite Toxoplasma gondii. Together, these results thoroughly uncover the dynamic reprogramming of macrophage nucleotide metabolism upon classical activation and elucidate the regulatory mechanisms and functional significance of such reprogramming.