This study probed the effects of Deshipu stachyose granules (DSG), a novel oligosaccharide preparation (55.3% stachyose, 25.8% raffinose, and 9.7% verbascose), on gut microbiota and constipation in mice. Mice were administered intragastrically without or with DSG (0.42, 0.83, and 2.49 g/kg bw), and feces were collected after 14 days of treatment and subjected to classical microbiological assays. Selective index (SI) and prebiotic index (PI) were incorporated to evaluate the prebiotic effect. DSG at 0.83 g/kg bw scored the highest SI and PI scores, thus supporting a strong prebiotic role. In addition, the impact of DSG (0.42, 0.83, and 1.68 g/kg bw) on defecation function of constipated mice was determined. Ink propulsion rate in the small intestine was significantly improved by DSG treatment. DSG supplementation also distinctly increased the weight and number of black feces within 5 h and evidently shortened the defecating time of first black feces, as compared with the constipation control mice. All of these findings indicate that DSG may promote the growth of beneficial intestinal bacteria and inhibit pathogenic bacteria and also facilitate intestinal peristalsis and fecal excretion, thereby enhancing intestinal health and relieving constipation.