Objective: To investigate the influence of antibiotics, bowel preparation, and food intake on gut bacteria of rats. Methods: Sixty SD rats were randomly divided into the control group, single factor treatment group (containing 3 subgroups: antibiotics group, bowel preparation group and fasting group), two factors treatment group (containing 3 subgroups: antibiotics + bowel preparation group, bowel preparation + fasting group and antibiotics + fasting group), three factors treatment group (antibiotics + bowel preparation + fasting group) and surgical treatment group (containing 2 subgroups: bowel preparation + antibiotics + operation + free diet group and bowel preparation + antibiotics + operation + postoperative fasting group). All groups were treated according to their predefined protocols for 4 days, and on the 5th day, all rats were killed by cervical dislocation and the stools taken from ileocecal junction under sterile conditions were used for bacterial culture and count. Results: The colibacillus number markedly increased, bifidobacterium and lactobacillus number, and bifidobactirium/colibacillus ratio markedly decreased, when the single factor treatment group was compared with the control group, the two factors treatment group was compared with the single factor treatment group, or the three factors treatment group was compared with the two factors treatment group respectively, and all the differences had statistical significances (all P<0.05). Furthermore, the above changes of gut flora in subgroups with antibiotics treatment were more evident than those in the subgroups of the same group without antibiotics treatment (all P<0.05). The colibacillus number was moderately reduced, and bifidobacterium and lactobacillus number in the free diet group was moderately increased compared to the postoperative fasting group, and the difference was statistically significant (P<0.05). Conclusion: Antibiotics, bowel preparation and fasting can all cause intestinal dysbacteriosis in rats, among which antibiotics have the greatest impact. Furthermore, the more numerous the treatment factors, the greater the influence. Early resumption of food intake has less influence on intestinal flora than postoperative fasting.