This study aimed to explore the effects of different levels of duck oil diglyceride and vitamin D₃ on colonic flora, spleen peroxisome proliferators activated receptor γ (PPARγ) mRNA expression and colonic tissue development of colitis rats. A total of 100 rats were randomly divided into model group and control group, each control group contained 4 replicates, each model group contained 16 replicates, and each replicate contained 5 rats; rats in control group free drank distilled water, rats in model group free drank 5% dextran sulfate sodium, and the modeling test for consecutive lasted for 7 days. After the successful molding, selected 10 normal rats as a control group (group Ⅰ), then selected 70 rats of successful molding randomly divided into 7 group, which were model group (group Ⅱ) and experimental groups (groups Ⅲ to Ⅷ); each group contained 2 replicates and each replicate contained 5 rats. A 2×3 cross factor analysis experiment design was adopted, the addition levels of duck oil diglyceride were 2.5,5.0 and 10.0 mL/kg, respectively, and the addition levels of vitamin D₃ was 12.5 and 25.0 μg/kg, respectively. The experiment lasted for 3 weeks. The results showed as follows:1) the interaction of duck oil diglyceride and vitamin D₃ had significant effects on Ace index and Chao1 index (P<0.05), compared with the model group, the operation classification unit (OTUs) number, Ace index and Chao1 index of experimental groups were significantly increased (P<0.05), the OTUs number, Ace index and Chao1 index of 5.0 mL/kg duck oil diglyceride and 12.5 μg/kg vitamin D₃ group were the highest, and the microbial species abundance was higher than other five experimental groups. 2) The principal component analysis figure showed that the 5.0 mL/kg duck oil diglyceride and 12.5 μg/kg vitamin D₃ group was closest to the control group, with the highest microbial similarity. 3) Compared with the model group, the proportions of Firmicutes, Proteobacteria, Gammaproteobacteria, Shigella, Prevotella and Allobaculum of experimental groups were significantly decreased (P<0.05), the proportions of Bacteroidetes and Bacteroides were significantly increased (P<0.05), and the Clostridia proportion had no significant change (P<0.05). 4) The interaction of duck oil diglyceride and vitamin D₃ had significant effects on villus height and crypt depth (P<0.05); compared with the model group, the villus height and villus height/crypt depth of experimental groups were significantly increased (P<0.05), and the crypt depth was significantly decreased (P<0.05). 5) The interaction of duck oil diglyceride and vitamin D₃ had significant effects on spleen PPARγ mRNA expression (P<0.05); compared with the model group, the spleen PPARγ mRNA expression of experimental groups (except 10.0 mL/kg duck oil diglyceride and 25.0 μg/kg vitamin D₃ group) was significantly increased (P<0.05), and the spleen PPARγ mRNA expression of 5.0 mL/kg duck oil diglyceride and 12.5 μg/kg vitamin D₃ group was the highest. 6) The spleen PPARγ mRNA expression was significantly positively correlated with villus height and illus height/crypt depth (P<0.01), and negatively correlated with crypt depth (P<0.01). In conclusion, different levels of duck oil diglyceride and vitamin D₃ can improve the microbial structure of colon flora of colitis rats, increase the proportion of beneficial microorganisms, promote the development of colon intestinal tract, up-regulate the spleen PPARγ mRNA expression, and have regulatory effect on intestinal development. The combination proportion of 5.0 mL/kg duck oil diglyceride and 12.5 μg/kg vitamin D₃ is the optimal combination.