Abstract Tumor cells alter their metabolism to meet their demand for macromolecules and support a high rate of proliferation as well as cope with oxidative stress. The transcription factor yin yang 1 (YY1) is upregulated in various types of tumors and is crucial for tumor cell proliferation and metastasis. However, its role in tumor cell metabolic reprogramming is poorly understood. Here, we show that YY1 alters tumor cell metabolism by activating glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway. By stimulating the pentose phosphate pathway, YY1 enhanced production of nucleotides and DNA synthesis, decreased intracellular reactive oxygen species levels, and promoted antioxidant defense by supplying increased reducing power in the form of NADPH. Importantly, YY1-mediated regulation of the pentose phosphate pathway in tumor cells occurred not through p53, but rather through direct activation of G6PD transcription by YY1. Regulation of pentose phosphate pathway activity through G6PD was strongly related to YY1-induced proliferation of tumor cells and tumorigenesis. Together, our results describe a novel role for YY1 in regulating G6PD in a p53-independent manner, which links its function in tumorigenesis to metabolic reprogramming in tumor cells. Significance: This study reveals a novel role for YY1 in regulating G6PD and activating the pentose phosphate pathway, linking its function in tumorigenesis to metabolic reprogramming. Cancer Res; 78(16); 4549–62. ©2018 AACR.