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Rehmanniae Radix (RR), the roots of Rehmannia glutinosa, is a popular Chinese materia medica. Both dried and prepared RR are used in Chinese prescriptions, but have totally different clinical purposes. A partially purified oligosaccharide from PRR (OPRR) was used to investigate its effect on oral starch tolerance test (OSTT) in fasted normal ICR mice, oral glucose tolerance test (OGTT) in diabetic db/db mice and gluconeogenesis in H4IIE cells. High-performance liquid chromatography (HPLC) served as a chemical analytical platform. Results showed OPRR treatment lowered postprandial glucose level and intestinal α-glucosidase activity in normal mice, and reversed glucose intolerance in diabetic db/db mice. In vitro study showed OPRR inhibited hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expressions in H4IIE cells. These results suggested that OPRR effectively decreased postprandial glucose level and intestinal α-glucosidase activity, and reversed glucose intolerance and inhibited gluconeogenesis. These findings suggest that OPRR may have a beneficial effect for diabetic hyperglycemia.