Objective This study aims to investigate the effects of high-fat diet rich in perilla oil on the insulin sensitivity-related gene expression in skeletal muscle in insulin resistant rats. Methods The insulin resistant(IR) rat models were randomly divided into 2 groups, including high fat group(HF) and perilla oil(PO) intervention group fed with 20% substitution of lard energy in the HF. The insulin sensitivity of rats was measured after 4 weeks. The α-linolenic acid(ALA) content of PO in the rat plasma were analyzed by gas chromatograph. Real-time PCR was applied to measure glucose transporter 4(GLUT4) and insulin receptor substrate-1(IRS-1) mRNA, and Western blot assay was used for detecting the expression of GLUT4 and IRS-1 in the skeletal muscle. Results At the gene and protein levels, PO remarkably reduced the level of IRS-1 and upregulated the level of GLUT4 with increasing intake of ALA and serum ALA content in IR rats. The results of hyperinsulinemic-euglycemic clamp test showed no significant difference between the two groups. Conclusions The results of our study suggest that consumption of n-3 PUFA at levels that can typically be found in the diet fed to IR rats in the form of ALA(0.556 g/d) may not improve insulin sensitivity, even though regulating the expression of GLUT4 and IRS-1 in the skeletal muscle.