Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLnA) have been known to have several health-promoting effects. The aim of this study was to introduce a novel structured lipid (SL) including both CLA and CLnA (cis9, trans11, cis13-18:3, punicic acid) into one triacylglycerol (TAG) molecule through enzymatic interesterification and investigate its effect on body weight, liver and serum lipids in Wistar rats. CLA oil, pomegranate seed oil (as a rich source of CLnA) and soybean oil (as a negative control) were applied as other experimental oils. The rats were fed the oils at 1500 mg/kg body weight per day via oral gavage for 45 days. Gas chromatography analysis showed that SL included CLnA and CLA in roughly equal concentrations. The in vivo study revealed that SL had the greatest effect on the reduction in liver lipid weight (4.65 g/100g of liver) and liver TAG (13.28 mg/g) compared to soybean oil (8.7 g/100g and 18.8 mg/g, respectively). High density lipoprotein cholesterol (HDL-C) in the serum of rats which were fed CLA oil significantly (p < 0.05) increased (from 0.95 to 1.14 mmol/l). Pomegranate seed oil reduced low density lipoprotein cholesterol (LDL-C) and total cholesterol (about 40% and 24% reduction, respectively). A remarkable TAG reduction (p < 0.05) was observed in all treated rats.