The purpose of this study is to explore the glycolytic remodeling under high-selenium (Se) stress. Three groups of male C57BL/6J mice were fed on diets with different Se contents (0.03, 0.15, and 0.30 mg Se/kg). Glucose tolerance test (GTT) and insulin tolerance test (ITT) were measured at the third month. Mice were killed at the fourth month. Plasma, liver, and muscle tissues were fetched for biochemistry and Se analysis. The expressions of insulin signaling pathway (PI3K-AKT-mTOR), glutathione peroxidase 1 (GPX1), selenoprotein N (SELENON), 3-phosphoglycerate dehydrogenase (PHGDH), serine hydroxymethyltransferases 1 (SHMT1), 5,10-methylenetetrahydrofolate reductase (MTHFR), and methionine synthase (MS) were analyzed by western blotting (WB) in liver and muscle tissues. The results of GTT and ITT showed that glucose tolerance and insulin tolerance were both abnormal in the 0.03 mg Se/kg and 0.3 mg Se/kg groups. Se concentrations in plasma, liver, and muscle of 0.03 mg Se/kg group were significantly lower than that of 0.15 mg Se/kg and 0.30 mg Se/kg groups (p < 0.05 or p < 0.01). The expressions of P-Akt (Thr-308) in muscle (p < 0.05) and PI3K and mTOR in liver (p < 0.001) of 0.30 mg Se/kg group were downregulated. The expressions of GPX1 in liver and muscle (p < 0.05 and p < 0.001), SELENON in muscle (p < 0.05), PHGDH in liver and muscle (p < 0.05), and SHMT1 (p < 0.05), MTHFR (p < 0.001), and MS (p < 0.001) in muscle of 0.3 mg Se/kg group were upregulated. The de novo serine synthesis pathway (SSP) was found to be activated in liver and muscle tissues of mice with a high-Se diet for the first time.