Excessive sugar intake including high-fructose corn syrup (HFCS) is implicated in the rise of obesity, insulin resistance and non-alcoholic fatty liver disease. Liver glycogen synthesis is influenced by both fructose and insulin signaling. Therefore, the effect of HFCS on hepatic glycogenesis was evaluated in mice feeding ad-libitum. Using deuterated water: the fraction of glycogen derived from triose-P sources, Krebs cycle substrates, and direct pathway + cycling, was measured in 9 normal-chow fed mice (NC) and 12 mice fed normal chow plus a 55% fructose/45% glucose mix in the drinking water at 30% w/v (HFCS-55). This was enriched with [U-13C]fructose or [U-13C]glucose to determine the contribution of each to glycogenesis. For NC, direct pathway + cycling, Krebs cycle, and triose-P sources accounted for 66 ± 0.7%, 23 ± 0.8% and 11 ± 0.4% of glycogen synthesis, respectively. HFCS-55 mice had similar direct pathway + cycling (64 ± 1%) but lower Krebs cycle (12 ± 1%, p < 0.001) and higher triose-P contributions (24 ± 1%, p < 0.001). HFCS-55-fructose contributed 17 ± 1% via triose-P and 2 ± 0% via Krebs cycle. HFCS-55-glucose contributed 16 ± 3% via direct pathway and 1 ± 0% via Krebs cycle. In conclusion, HFCS-55 supplementation resulted in similar hepatic glycogen deposition rates. Indirect pathway contributions shifted from Krebs cycle to Triose-P sources reflecting HFCS-55-fructose utilization, while HFCS-55-glucose was incorporated almost exclusively by the direct pathway.