Folate metabolism is intricately linked to purine de novo synthesis through the incorporation of folate-derived one-carbon units into the purine scaffold. By investigating chemical and genetic dependencies caused by mutations in methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1), we discovered a key role for Nudix hydrolase 5 (NUDT5) in regulating purine de novo synthesis. Genetic depletion and selective chemical degradation showed that a scaffolding role, rather than NUDT5 enzymatic activity, was causing this phenotype. NUDT5 interacted with phosphoribosyl pyrophosphate amidotransferase (PPAT), the rate-limiting enzyme of purine de novo synthesis, to repress the pathway in response to increased purine abundance. Through this mechanism, loss of NUDT5 mediates resistance to purine analogs in cancer treatment and prevents adenosine toxicity in MTHFD1 deficiency.