Objective To investigate the effect of vitamin D ( VD) on intestinal flora in spontaneously diabetic rats. Methods Zucker diabetic fatty rats ( ZDF rats) were randomised to control (Con) group, VD control (VD) group,model ( T2DM) group, and VD intervention (VD+T2DM) group. Fasting blood glucose profiles and oral glucose tolerance levels were determined in rats of each group. 16S rDNA sequencing was used to assess changes in rat intestinal flora. OTU analysis (Venn diagram) , α diversity analysis ( chao1, observed species, PD whole tree, and shannon and simpson) , β diversity analysis ( principal coordinate analysis ( PCoA) ) , flora structure, and colony species variability analysis ( linear discriminant analysis and influence factor ( LEfSe) analysis) were also performed. Results VD intervention significantly improved fasting blood glucose levels and insulin resistance in T2DM rats ( P< 0. 05) . α diversity showed no significant differences in chao1, observed species, PD whole tree, and shannon and simpson indices between T2DM and VD+T2DM groups (P>0. 05) . β diversity analysis showed that the VD+T2DM group had more species similarity to the Con group than the T2DM group. The dominant bacteria of rat intestinal flora in each group were significantly different. In comparison to the T2DM group, the VD+T2DM group showed a decrease in abundance of Bacteroidetes and increases in abundances of Firmicutes and Clostridium XIVa. Conclusions VD improves fasting glucose elevation and insulin resistance in T2DM rats. VD improves the structure of intestinal flora, decreases Bacteroidetes, and elevates Firmicutes and Clostridium XIVa abundances in T2DM rats.