Dendrobium officinale is a traditional Chinese medicine for treating gastrointestinal diseases by nourishing “Yin” and thickening the stomach lining. To study whether D. officinale endophytes can colonize the intestinal tract and regulate gut microbiota in mice, we used autoclave steam sterilizing and 60Co-γ radiation to eliminate D. officinale endophytes from its juice. Then, high-throughput ITS1-ITS2 rDNA and 16S rRNA gene amplicons were sequenced to analyze the microbial community of D. officinale endophytes and fecal samples of mice after administration of fresh D. officinale juice. Sterilization of D. officinale juice by autoclaving for 40 min (ASDO40) could more effectively eliminate the D. officinale endophytes and decrease their interference on the gut microbiota. D. officinale juice could increase beneficial gut microbiota and metabolites including short-chain fatty acids. D. officinale endophytes Pseudomonas mosselii, Trichocladium asperum, Titata maxilliformis, Clonostachys epichloe, and Rhodotorula babjevae could colonize the intestinal tract of mice and modulate gut microbiota after oral administration of the juice for 28 days. Thus, the regulatory effect of D. officinale juice on gut microbiota was observed, which provides a basis for inferring that D. officinale endophytes might colonize the intestinal tract and participate in regulating gut microbiota to treat diseases. Thus, this study further provides a new approach for the treatment of diseases by colonizing plant endophytes in the intestinal tract and regulating gut microbiota.