<p>Glycolysis is a ubiquitous energy-supply process that occurs in virtually all living cells. The phenomenon of aerobic glycolysis, known as the Warburg effect, is observed and considered as a hallmark of tumorigenesis. However, the roles and unresolved mechanisms of glycolytic enzymes in cancer progression remain incompletely understood. In this review, we synthesize emerging knowledge on the non-glycolytic (“moonlighting”) functions of key glycolytic enzymes in tumorigenesis, which are independent of their canonical catalytic activity and enable them to govern diverse cellular processes beyond energy metabolism. We also discuss their potential as therapeutic targets and establish a conceptual framework for understanding how these moonlighting activities govern tumor progression, thereby supporting the development of novel anticancer therapies.</p><p><?noindent??><b>Clinical trial number</b></p><p><?noindent??>Not applicable.</p>