The current study found that deep-frying process led to an increased content of oxidized triacylglycerols in canola oil, 3.5 times higher than that of fresh canola oil (not used for frying). A rat model was then used to study the effect of the consumption of oxidized oil on blood lipid compositions and investigate the mechanism involved in lipids metabolism in the liver of rats with resistant starch (RS) intervention. Studies involving animals revealed that the consumption of deep-fried oil (DO group) significantly reduced the level of both triglycerides and high-density lipoprotein cholesterols (P < 0.05) in the serum, indicating that lipid biosynthesis was impaired. However, the supplementation of RS in the DO-containing diet (DO-RS group) attenuated the abovementioned status. The further study found that Insig expression was down-regulated with an increased mRNA expression of PPARα, together with reduced expressions of SREBP1 and downstream lipogenesis-associated genes in the rats of the DO group. In contrast, RS supplementation up-regulated Prkag2 (an AMPK related gene) and Insig-1/2 expressions in the DO-RS group compared to that in the DO group. The activation of the Insig pathway might be one of the key regulators for attenuating the impaired lipid biosynthesis induced by the oxidized fat following RS intervention.