Both nutritive and non-nutritive sweeteners may influence energy and glucose metabolism differently. The hypothesis that sucrose, fructose, aspartame, and sucralose intake differently modulate energy and glucose metabolism was tested in an estrogen-deficient animal model. At 30 min after giving aspartame and sucralose (10 mg/kg body weight), an oral glucose tolerance test (OGTT) was conducted with glucose, sucrose, and fructose in ovariectomized (OVX) rats. After OGTT, they were continuously fed high fat diets including either 10% corn starch (Control), 10% sucrose (Sucrose), 10% fructose (Fructose), 0.05% aspartame + 9.95% starch (Aspartame) or 0.05% sucralose + 9.95% starch (Sucralose) for 8 week. During 30 min after acute administration of aspartame and sucralose, serum glucose concentrations increased despite slightly increased serum insulin levels before glucose infusion. However, glucose tolerance was not significantly different among the groups. In chronic study, serum glucose concentrations were lowest and insulin highest at the overnight-fasted state in Aspartame and Sucralose. Postprandial serum glucagon-like peptide-1 (GLP-1) and insulin levels were higher in Aspartame and Sucralose than Control. Hepatic insulin signaling (pAkt → pGSK-3β) and phosphoenolpyruvate carboxykinase (PEPCK) expression were lower in Sucralose and Aspartame than the Fructose. Serum acetate levels produced by gut microbiota were higher were lower in the fructose group than Aspartame and Sucralose groups. In conclusion, aspartame and sucralose with a meal might be preferable sweeteners to fructose and sucrose in estrogen deficient rats, and possibly post-menopausal women; however, this needs to be confirmed in human studies.