Glycogenolysis and gluconeogenesis are sensitive to nutritional state and their balances are disrupted in the liver by different pathological states. The regulation of the balance between glucose production and synthesis of glycogen continues to be a matter of investigation because of its implications in diseases and its value for therapeutics. We used the gluconeogenic precursor dihydroxyacetone (DHA) to study glycogen synthesis in isolated rat hepatocytes under gluconeogenic conditions. We used a glycogen phosphorylase (GP) inhibitor, 2-deoxy-2-fluoro-α-D-glucopyranosyl fluoride (F2Glc) to prevented GP from breaking up glycogen into glucose subunits and had a pool of glucose in the medium. Therefore, we evaluated the contribution of DHA as a unique source of carbohydrates on glycogen metabolism. We showed that DHA increased G6P levels that induced both GS activation and its translocation and, thus, an increment of glycogen deposition in a similar way as glucose did.