Although conjugated linoleic acid (CLA) has been shown to have anti-obesity properties, the effect and mechanism of CLA in alleviating glycolipid metabolism disorders remains unclear. In this work, it was observed that rats fed a high-fat diet (HFD) had lower body weight and body fat levels after 9 weeks of low-dose and high-dose CLA interventions. The results of blood biochemical indices showed that CLA significantly reduced the levels of total cholesterol, triglycerides, fasting blood glucose and insulin. Additionally, high-dose CLA could restore the intestinal microbiota composition, including increasing the relative abundances of short-chain fatty acid (SCFA)-producing microbiota, such as Dubosiella, Faecalibaculum and Bifidobacterium; decreasing the relative abundances of Enterococcus and Ruminococcus_2; and increasing the content of SCFAs in feces and serum. Further analysis showed that high-dose CLA could increase the expression levels of Insr, Irs-2, Akt and Glut4 in the liver tissue of HFD-induced obese rats. Consistently, high dose of CLA could reversibly improve the downregulation of INSR, AKT, PI3K and GLUT4 protein expression caused by HFD and reverse the decline in AKT phosphorylation levels. Correlation clustering analysis with a heatmap showed that the changes in specific microbiota induced by high-dose CLA were correlated with changes in obesity-related indices and gene expression. The molecular docking analysis showed that the molecular docking of SCFAs with the IRS-2, AKT and GLUT4 proteins had high linking activity. The results supported that CLA can alleviate glycolipid metabolic imbalances associated with obesity by altering the intestinal microbiota to induce the production of SCFAs and thereby activate the INSR/IRS-2/AKT/GLUT4 pathway. This study supports CLA may be preferentially used by the intestinal microbiota of the host to promote its health.