Fructose‐2, 6‐bisphosphate is an important intracellular biofactor in the control of carbohydrate metabolic fluxes in eukaryotes. It is generated from ATP and fructose‐6‐phosphate by 6‐phosphofructo‐2‐kinase and degraded to fructose‐6‐phosphate and phosphate ion by fructose‐2, 6‐bisphosphatase. In most organisms these enzymatic activities are contained in a single polypeptide. The reciprocal modulation of the kinase and bisphosphatase activities by post‐translational modifications places the level of the biofactor under the control of extra‐cellular signals. In general, these signals are generated in response to changing nutritional states, therefore, fructose‐2, 6‐bisphosphate plays a role in the adaptation of organisms, and the tissues within them, to changes in environmental and metabolic states. Although the specific mechanism of fructose‐2, 6‐bisphosphate action varies between species and between tissues, most involve the allosteric activation of 6‐phosphofructo‐1‐kinase and inhibition of fructose‐1, 6‐bisphosphatase. These highly conserved enzymes regulate the fructose‐6‐phosphate/fructose‐1, 6‐bisphosphate cycle, and thereby, determine the carbon flux. It is by reciprocal modulation of these activities that fructose‐2, 6‐bisphosphate plays a fundamental role in eukaryotic carbohydrate metabolism.