Vitamin D (VD) is associated with the release of leptin, which promotes fat mobilization in the body. Diacylglycerol can reduce triacylglycerol formation. This study evaluated the effects of administration of VD and duck oil diacylglycerol on blood lipids, liver lipid metabolism and gut microflora in rats fed a high-fat diet. A total of 75 rats were allocated randomly to five treatments: non-obesity (NO), obesity control (OC), diacylglycerol:10 mL per kg body weight (DG10), VD:12.5 μg per kg body weight (VD12.5), and diacylglycerol + VD: 10 mL/d + 12.5 μg per kg body weight (DG10VD12.5). Rats in NO group was fed the diet containing 7% soybean oil, while the other four groups were fed a high-fat (3.3% soybean oil and 30.1% lard). VD and diacylglycerol were orally dosed once per day. The administration of VD+diacylglycerol significantly reduced the body weight gain and fat content. The change of fat deposit was accompanied with the reduction in serum lipids, the increase in the activity of enzymes involved in lipid metabolism in the liver, the suppression of fatty acid synthase and the enhance of carnitine fatty acyl transferase Ⅰ (CPT1) expression. The administration of VD and diacylglycerol also significant modulated the bacterial diversity profile in the cecum towards that in non-obesity rats. Those results suggest that the administration of VD and diacylglycerol could have significant roles in reducing fat deposit and altering gut microflora in rats fed the high-fat diet.