Hyperinsulinemia is widely thought to be a compensatory response to insulin resistance, whereas its potentially causal role in the progression of insulin resistance remains to be established. Here, we aimed to examine whether hyperinsulinemia could affect the progression of insulin resistance in Zucker fatty diabetic (ZDF) rats. Male ZDF rats, at 8 weeks of age, were fed a diet ad libitum (AL) or DR of either 15% or 30% from AL feeding over 6 weeks. Insulin sensitivity was determined by hyperinsulinemic-euglycemic clamp. ZDF rats in AL group progressively developed hyperglycemia and hyperinsulinemia by 10 weeks of age, and then plasma insulin rapidly declined to near normal levels by 12 weeks of age. Compared with AL group, DR groups showed delayed onset of hyperglycemia and persistent hyperinsulinemia, leading to weight gain and raised plasma triglyceride and free fatty acid by 14 weeks of age. Notably, insulin sensitivity was significantly reduced in DR group rather than AL group, and was inversely correlated with plasma levels of insulin and triglyceride, but not glucose. Moreover, enhanced lipid deposition and up-regulation of genes involved in lipogenesis were detected in liver, skeletal muscle and adipose tissues of DR group rather than AL group. Alternatively, continuous hyperinsulinemia induced by insulin pellet implantation produced a decrease in insulin sensitivity in ZDF rats. These results suggest that chronic hyperinsulinemia may lead to the progression of insulin resistance under DR conditions in association with altered lipid metabolism in peripheral tissues in ZDF rats.