Metabolic reprogramming is a hallmark of cancers crucial for fulfilling the needs of energy, building blocks, and antioxidants to support tumor cells' rapid proliferation and to cope with the harsh microenvironment. Pre-B-cell leukemia transcription factor 3 (PBX3) is a member of the PBX family whose expression is up-regulated in various tumors, however, whether it is involved in tumor cell metabolic reprogramming remains unclear. Herein, we report that PBX3 is a positive regulator of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). PBX3 promoted transcriptional activity in tumor cells by binding directly to its promoter, leading to PPP stimulation and enhancing the production of nucleotides and NADPH, a crucial reductant, thereby promoting nucleic acid and lipid biosynthesis while decreasing intracellular reactive oxygen species levels. The PBX3/G6PD axis also promoted tumorigenic potential and . Collectively, these findings reveal a novel function of PBX3 as a regulator of G6PD, linking its oncogenic activity with tumor cell metabolic reprogramming, especially PPP. Furthermore, our results suggested that PBX3 is a potential target for metabolic-based anti-tumor therapeutic strategies.