We investigated the physiological activity of an oil rich in γ-linolenic acid of evening primrose origin (containing 42.6% γ-linolenic acid) affecting hepatic fatty acid metabolism, and serum lipid levels in genetically hyperlipidemic mice deficient in apolipoprotein E expression. Male apolipoprotein E-deficient mice (BALB/c.KOR/StmSlc-Apoe ) were fed experimental diets containing 100 g/kg of palm oil (saturated fat), safflower oil (rich in linoleic acid), γ-linolenic acid oil (rich in γ-linolenic acid), or fat mixtures composed of safflower and γ-linolenic acid oils (65:35 and 30:70, w/w) for 20 days. γ-Linolenic acid oil, compared with palm and safflower oils, strongly and dose-dependently increased the activity and mRNA levels of hepatic fatty acid oxidation enzymes. In general, safflower and γ-linolenic acid oils, compared with palm oil, reduced the activity and mRNA levels of lipogenic enzymes. However, these oils were equivalent in reducing the parameters of lipogenesis, excluding malic enzyme and pyruvate kinase. The diets containing safflower and γ-linolenic acid oils, compared with the palm oil diet, significantly decreased serum triacylglycerol and cholesterol levels. The decreases were greater with γ-linolenic acid oil than with safflower oil. γ-Linolenic acid oil exerted strong serum lipid-lowering effects in apolipoprotein E-deficient mice apparently through the changes in hepatic fatty acid metabolism.