We examined the effect of docosahexaenoic acid (DHA)-enriched structured lipids-diacylglycerol (SL-DG), which were synthesized using soybean oil (SO) and algae oil (AO), on hepatic lipid metabolism and the mRNA expression of genes involved in hepatic steatosis of C57BL/6J-Lep(ob/ob) compared to the SL-triacylglycerol (TG). The animals were fed a high-fat (10% lard and 10% test oils) and high-cholesterol (0.2% cholesterol) diet for 12 weeks. Mice fed SL-DG showed a lower total white adipose tissue weight and plasma triglyceride concentration than the SO group. Reduction of hepatic triglyceride content in the SL-DG group was related with the suppression of hepatic enzyme activities for fatty acid and triglyceride synthesis along with fecal triglyceride excretion compared to the SL-TG. SL-DG also lowered hepatic cholesterol levels by suppressing cholesterol regulating enzyme activity compared to the SO group. Moreover, SL-DG lowered the mRNA expressions of sterol regulatory element binding protein-1 and its target genes than TG-form oils (SO, AO and SL-TG) in the liver. Thus, the current results suggest that DHA-enriched SL-DG oil used in this study is beneficial for ameliorating hepatic steatosis in obese animal model by improving hepatic fatty acid and cholesterol metabolic enzyme activity and their gene expression.