Abstract: Objective To assess the effect of long-term high-fat diet on the expressions of insulin receptor substrates in the hippocampus and spatial learning and memory ability of obese rats. Methods A total of 100 4-week-old male SD rats were randomly divided into two groups and fed with common diet (CD group, n=40) or high-fat diet (HFD group, n=60) for 16 weeks. At 4, 8, 12, 16 and 20 weeks, 8 rats were randomly selected from each group for testing their spatial learning and memory function using Morris water maze. After the tests, the rats were sacrificed for measurement of the metabolic parameters and detection of the expressions of insulin receptor substrate-1 (IRS-1) and IRS-2 mRNAs in the CA1 region of the hippocampus. Results Compared with those in CD group, the rats in HFD group showed a prolonged escape latency, longer swimming distance, faster average swimming speed, and shorter stay in the platformat 12 weeks. In HFD group, the serum levels of total cholesterol, triglyceride, low-density lipoprotein cholesterol, and fasting insulin were all significantly increased (P<0.05) and the level of high-density lipoprotein cholesterol decreased (P<0.01) in comparison with those in CD group at each of the time points. No significant difference was found in fast glucose levels between the two groups (P>0.05), but the expressions of IRS-1 and IRS-2 mRNAs were significantly decreased in HFD group at 12 weeks (P<0.05). Conclusion In obese rats, long-term feeding with high-fat diet leads to insulin resistance, which interferes with hippocampal expression of insulin receptor substrates and insulin metabolism to cause impairment of the cognitive function and accelerate cognitive deterioration.