Background: Short bowel syndrome (SBS) may result in nutrient malabsorption, intestinal dysfunction, and even complete intestinal failure, thereby compromising the quality of life and psychological status of patients. Animal models may help understand the mechanism involved in SBS, and improve its prognosis. Objective: To study changes in the behavioral pattern in a rat model of type III SBS, and it relationships with gut flora. Methods: A total of 20 male adult Sprague-Dawley (SD) rats, each weighing about 230 g, were randomly divided into an SBS group and a control group, with 10 rats in each group. Type III SBS model was established in the SBS group, while sham operation was adopted in the control group. All rats were kept for two weeks after surgery. Results: Massive small bowel resection resulted in significantly decreased weights of rats in SBS group, while control rats had marked increases in weight. Results of open-field test and sucrose preference test conducted two weeks postoperatively showed significantly lower sucrose preference index and lower frequency of horizontal and vertical movements in the SBS group (p < 0.05). The results of 16S ribosomal DNA (rDNA) sequencing in intestinal flora showed lower abundance of Firmicutes and Bacteroidetes and higher abundance of the Proteobacteria and Spirochaetes in the SBS group, although the differences were not statistically significant. Conclusions: Type III SBS rats exhibited significant depression/anxiety-like behavioral abnormalities at two postoperative weeks. This may be related to changes in intestinal flora after intestinal cut-off and anastomosis. However, there is need for further studies in this regard.