Dynamic partitioning of de novo purine biosynthetic enzymes into multienzyme compartments, purinosomes, has been associated with increased flux of de novo purine biosynthesis in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells. We have investigated the functional role of AMP-activated protein kinase (AMPK) in the regulation of de novo purine biosynthesis because of its regulatory action on lipid and carbohydrate biosynthetic pathways. Using pharmacological AMPK activators, we have monitored subcellular localizations of six pathway enzymes tagged with green fluorescent proteins under time-lapse fluorescence single-cell microscopy. We revealed that only one out of six pathway enzymes, formylglycinamidine ribonucleotide synthase (FGAMS), formed spatially distinct cytoplasmic granules after treatment with AMPK activators, indicating the formation of single-enzyme self-assemblies. In addition, subsequent biophysical studies using fluorescence recovery after photobleaching showed that the diffusion kinetics of FGAMS were slower when it localized inside the self-assemblies than within the purinosomes. Importantly, high-performance liquid chromatographic studies revealed that the formation of AMPK-promoted FGAMS self-assembly caused the reduction of purine metabolites in HeLa cells, indicating the downregulation of de novo purine biosynthesis. Collectively, we demonstrate here that the spatial sequestration of FGAMS by AMPK is a mechanism by which de novo purine biosynthesis is downregulated in human cells.