Abstract: Objective: To investigate the effect of Dendrobium nobile Lindl extract on improvement of constipation and intestinal Lactobacillus diversity of mice. Methods: The mice with constipation induced by in compound diphenoxylate were given the high-dose (300 mg/kg), middle-dose (100 mg/kg), low-dose (30 mg/kg) of Dendrobium nobile Lindl extract, respectively. The defecation function and intestinal propulsive rate in mice were observed. Using Lactobacillus-specific primer and the microbial genomic DNA extracted from the fecal samples, the ARDRA analysis of Lactobacillus diversity was accomplished after PCR amplification and enzyme digestion. Results: After intragastric administration, the defecation time of the first black stool decreased significantly (P<0.01). The number of fecal particles was increased, and the fecal water content increased significantly (P<0.01). The intestinal propulsive rate increased significantly in the middle-dose group and the high-dose group (P<0.05). The diversity of Lactobacillus increased after intragastric administration by Dendrobium nobile Lindl extract, and the evenness of Lactobacillus in the intestine was more stable in the high-dose group. Conclusion: It showed that Dendrobium nobile extract had an improvement effect on the symptoms of constipation by increasing the fecal water content, promoting the intestinal peristalsis and regulating the diversity of intestinal Lactobacillus in mice.