[Abstract] Objective To investigate the effect of vitamin D (VD) on intestinal flora in spontaneously diabetic rats. Methods Zucker diabetic fatty rats (ZDF rats) were randomly divided into control group (Con), VD control group (VD), model group (T2DM) and VD intervention group (VD+T2DM). Fasting blood glucose profile and oral glucose tolerance level were detected in rats in each group. The 16S rDNA sequencing technology was used to detect the changes in rat intestinal flora, and OTU analysis (Venn diagram), Alpha diversity analysis (chao1, observed species, PD whole tree, shannon and simpson), Beta diversity analysis [principal coordinate analysis (PCoA)], flora structure and colony species variability analysis [linear discriminant analysis and influence factor (LEfSe) analysis]. Results VD intervention significantly improved fasting blood glucose levels and insulin resistance in T2DM rats (P<0.05).Alpha diversity results showed no significant differences in chao1, observed species, PD whole tree, shannon and simpson indices between T2DM and VD+T2DM groups (P>0.05) ; Beta diversity analysis showed that the VD+T2DM group had more species similarity with the Con group compared with the T2DM group. The dominant bacteria of rat intestinal flora in each group were significantly different; compared with the T2DM group, the VD+T2DM group showed decreased abundance of Bacteroidetes and increased abundance of Firmicutes and Clostridium XIVa. Conclusion VD improved fasting glucose elevation and insulin resistance in T2DM rats; VD improved the structure of intestinal flora, decreased Bacteroidetes, and elevated Firmicutes and Clostridium XIVa abundance in T2DM rats.