Dietary fructose can benefit or hinder glycemic control, depending on the quantity consumed, and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that²H enrichment of glycogen positions 5 and 2 from deuterated water (²H₂O) informs direct and indirect pathway contributions to glycogenesis in naturally feeding rats. Inclusion of position 6_S²H enrichment data allows indirect pathway sources to be further resolved into triose phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC) and six rats that had fed on SC plus 35% sucrose in their drinking water (HS). After 2 wk, hepatic glycogenesis sources during overnight feeding were determined by²H₂O administration and postmortem analysis of glycogen²H enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 μmol/g dry wt HS vs. 578 ± 76 μmol/g dry wt SC), triose phosphate contributions were significantly higher for HS compared with SC (382 ± 61 vs. 87 ± 24 μmol/g dry wt, P < 0.01) and Krebs cycle inputs lower for HS compared with SC (110 ± 9 vs. 197 ± 32 μmol/g dry wt, P < 0.05). Analysis of plasma glucose²H enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose phosphate to plasma glucose levels in HS vs. SC. Hence, the²H enrichment distributions of hepatic glycogen and glucose from²H₂O inform the contribution of dietary fructose to hepatic glycogen and glucose synthesis.