Abstract: Objective To analyze the effect of conjugated linoleic acid (CLA) on glucose and lipid metabolism in obese diabetic (db/db) mice. Methods db/db mice were randomized for treatment with saline or CLA mixture administered intragastrically. The changes in body weight, dietary intake, water intake, oral glucose tolerance, triglyceride and total cholesterol were recorded after the treatments. HE staining and oil red O staining were used to assess liver pathologies and fatty acid content. The expression levels of PPARα, PPARγ, CD36, CHREBP and SREBP-1c were detected using real-time PCR and Western blotting. HepG2 cells were treated with CLA and linoleic acid and the expressions of PPARα, ACC, P-ACC, and CD36 were detected; the level of acetyl-CoA in the cell supernatant was detected using ELISA. Results CLA treatment obviously reduced the dietary and water intake of db/db mice, effectively reduced the body weight and decreased serum triglyceride and cholesterol levels (P<0.05). CLA significantly reduced fasting blood glucose, increased glucose tolerance, reduced the accumulation of lipid droplets in the liver and improved lipid metabolism in db/db mice. The mice showed significantly increased expression of PPARα (P<0.05) and lowered CD36 expression (P<0.001) in the liver after CLA treatment. Cellular experiments showed that CLA significantly up-regulated PPARα (P<0.001) and P-ACC and decreased the expression of CD36 (P<0.01). ELISA showed that acetyl-CoA was significantly up-regulated in the cells after CLA treatment (P<0.01). Conclusion The mixture of two conjugated linoleic acid isomers can reduce fasting blood glucose, increase glucose tolerance and improve glycolipid metabolism in db/db mice by enhancing the expression of PPARα, increasing P-ACC and inhibiting CD36 expression.