Glycolysis is a central metabolic pathway in all living organisms. Regulation of glycolysis is critical for stem cell differentiation because pluripotent stem cells meet their need for continuous self-renewal by using glycolysis to provide building blocks for the biosynthesis of nucleotides, peptides and lipids. While glycolysis is required for stemness, a shift towards oxidative phosphorylation accompanies lineage differentiation. However, the mechanisms underlying this metabolic rewiring remain to be better understood. We discovered that the catalytic activity of the glycolytic enzyme enolase 1 (ENO1) is regulated by RNAs through direct interaction, leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We further identified RNA ligands that specifically inhibit ENO1’s enzymatic activity in vitro and reduce glycolysis in cultured human cells and mESCs. ENO1’s association with RNA is altered by acetylation, with pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increasing ENO1’s RNA binding. Similarly, induction of differentiation leads to increased ENO1 acetylation, enhanced RNA binding and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or over-activate this regulation showed impaired germ layer differentiation. Our findings suggest that acetylation-driven RNA-mediated regulation of ENO1 is a physiological mechanism in the glycolytic control of stem cell differentiation. In fact, riboregulation may represent a more widespread principle of biological control with broad implications for our understanding of cellular differentiation, metabolic homeostasis and ageing, which we are now trying to uncover further.