Mitochondrial DNA (mtDNA) G-quadruplexes (G4s) have been proposed as potential regulators of energy metabolism. However, numerous aspects of this process remain elusive. In our quest, employing a chemical-genetic screening strategy, we successfully identified the JAK/STAT3 pathway as the primary regulatory mechanism controlling mtDNA G4 dynamics in hypoxic MCF-7 cells. Further proteomic analyses and associated investigations have shed light on the translocation of RelA, a member of the NF-κB family, to the mitochondria upon activation of the JAK/STAT3 pathway. Once localized, RelA directly binds to mtDNA G4s and facilitates their folding, consequently yielding escalated mtDNA instability, impaired mtDNA transcription, and ensuing mitochondrial dysfunction. Ultimately, this intricately orchestrated cascade disrupts the equilibrium of energy metabolism, favoring a more pronounced deviation towards glycolysis. This pioneering revelation introduces an unprecedented and pivotal component to the intricately interconnected framework harnessed by cancer cells, empowering them to undergo metabolic reprogramming and adapt to hypoxic environments.