Water-insoluble β-glucan has been reported to have beneficial effects on human health. However, no studies have thoroughly characterized the structure and function of water-insoluble β-glucan in oat bran. Thus, the structure and effect of water-insoluble β-glucan on weight gain and lipid metabolism in high-fat diet (HFD)-fed mice were analyzed. First, water-insoluble β-glucan was isolated and purified from oat bran. Compared with water-soluble β-glucan, water-insoluble β-glucan had higher DP3:DP4 molar ratio (2.12 and 1.67, respectively) and molecular weight (123,800 and 119,200 g/mol, respectively). Notably, water-insoluble β-glucan exhibited more fibrous sheet-like structure and greater swelling power than water-soluble β-glucan. Animal experiments have shown that oral administration of water-insoluble β-glucan tended to lower the final body weight of obese mice after 10 weeks treatment. In addition, water-insoluble β-glucan administration significantly improved the serum lipid profile (triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels) and epididymal adipocytes size. What is more, water-insoluble β-glucan reduced the accumulation and accelerated the decomposition of lipid in liver. In conclusion, water-insoluble β-glucan (oat bran) could alleviate obesity in HFD-fed mice by improving blood lipid level and accelerating the decomposition of lipid.