SRP309436 PRJNA706901 6-Phosphogluconate dehydrogenase (6PGD) a key checkpoint in reprogramming of Treg metabolism and function ellular metabolism has key roles in T cells differentiation and function. CD4+ T helper-1 (Th1), Th2 and Th17 subsets are highly glycolytic while regulatory T cells (Tregs) use glucose during expansion but rely on fatty acid oxidation for function. Upon uptake, glucose can enter pentose phosphate pathway (PPP) or be used in glycolysis. Here we showed that blocking 6- phosphogluconate dehydrogenase (6PGD) in the oxidative PPP resulted in substantial reduction of Tregs suppressive function and shift toward Th1, Th2 and Th17 phenotypes. These in turn improved anti-tumor responses and worsened the outcomes of colitis model. Metabolically, 6PGD deficient Tregs showed improved glycolysis and enhanced non-oxidative PPP to support nucleotide biosynthesis. These results uncover critical role of 6PGD in modulating Tregs plasticity and function, which qualifies it as a novel metabolic checkpoint for immunotherapy applications.