Lupin protein has been shown to reduce triglyceride and cholesterol concentrations in plasma of hypercholesterolemic growing and adult rats. However, the effect of lupin protein on lipid metabolism during pregnancy and lactation is unknown. Female rats were assigned to two groups and were fed a hypercholesterolemic diet containing either 200 g/kg lupin protein or casein during pregnancy until day 18 of lactation. Dams fed lupin protein had lower triglyceride concentrations in plasma (-55%) and higher triglyceride concentrations in liver (>2-fold) and milk (+81%) than dams fed casein (p < 0.05). The concentration of cholesterol in plasma, VLDL, LDL, and liver was markedly lower and the concentration of HDL cholesterol was higher in rats fed lupin protein than in rats fed casein (p < 0.05). Lupin protein induced a 2.6-fold increase of hepatic LDL receptor concentration compared to casein (p < 0.05), down-regulated genes involved in fatty acid oxidation in the liver, and up-regulated lipogenic genes in the mammary gland (p < 0.05). This study shows that lupin protein increases milk fat content and strongly modifies triglyceride and cholesterol metabolism by influencing the transcription levels of genes involved in fatty acid oxidation and synthesis and cholesterol homeostasis.