In vitro and in vivo studies have shown that punicic acid, a type of conjugated fatty acid and the main constituent of pomegranate seed oil (PSO), has anti-atherogenic effects. The present study aimed at determining the effect of PSO treatment on serum lipid profiles. This double-blind placebo-controlled randomised clinical trial included fifty-one hyperlipidaemic subjects, diagnosed according to National Cholesterol Education Program definition, and randomly assigned to the PSO and the control groups. The PSO and placebo groups received 400 mg PSO and placebo twice daily, respectively and were followed up for 4 weeks. Serum concentrations of lipids and lipoproteins were measured before and 4 weeks after intervention. Mean concentration of TAG and the TAG:HDL cholesterol (HDL-C) ratio were significantly decreased after 4 weeks in the PSO group as compared with baseline values (2·75 (sd 1·40) v. 3·45 (sd 1·56) mmol/l, P = 0·009 and 5·7 (sd 4·6) v. 7·5 (sd 5·0), P = 0·031, respectively). The treatment effect was statistically significant in the PSO group as compared with controls in diminution of cholesterol:HDL-C ratio (5·4 (sd 1·5) v. 5·9 (sd 1·4), P < 0·05) adjusted for baseline values. We found a mean difference for PSO v. placebo in HDL-C concentration (0·13 v. − 0·02 mmol/l) and cholesterol:HDL-C ratio ( − 0·42 v. 0·01, P < 0·05). Serum cholesterol, LDL cholesterol and glucose concentrations and body composition variables remained unchanged. It is concluded that administration of PSO for 4 weeks in hyperlipidaemic subjects had favourable effects on lipid profiles including TAG and TAG:HDL-C ratio.