Objective To prepare an irritable bowel syndrome with diarrhea ( IBS-D) mouse model by rectal perfusion with acetic acid combined with restraint stress, and to evaluate the feasibility of the model. Methods Forty-five male C57BL/ 6J mice were divided into a healthy control group (Group C), 3% acetic acid group (Group A), and 3% acetic acid combined with restraint stress group (Group A+R). The body weights and survival rates of the groups were compared before and after intervention, and the fecal water content, total intestinal transport time, and colonic pain threshold of each group were compared after intervention. The advantages and disadvantages of the two modeling method were then assessed. Pathological changes to the colons in Group C and Group A + R were observed. Fluorescein isothiocyanate-dextran 4 (FD4) was used to evaluate the integrity of the intestinal mucosa of mice in Group C, and the model with the best success rate was determined. To clarify the pathological characteristics of this modeling method, the serum contents of 5-HT and TNF-α were determined by ELISA, and the expression of 5-HTR3A in colon tissue was detected with immunohistochemistry. Results Compared with Group C, Group A+R had a significantly increased colonic pain threshold and fecal water content (P< 0.001). The total intestinal transport time was significantly shortened (P< 0.05), and the colonic pain threshold and fecal water content were significantly decreased in Group A ( P< 0.005); however, there was no significant difference in intestinal transport time between the groups (P> 0.05). Compared with Group C, Group A+C showed a significant increase in the content of plasma FD4 and serum 5-HT and TNF-α and the expression of 5-HTR3A in colon tissues (P< 0.05). Conclusions Compared with 3% acetic acid, treatment with 3% acetic acid combined with restraint stress more convincingly simulated the local intestinal symptoms and related pathological changes seen in IBS-D patients.