Functional properties and physiological effects have been described for the lupin, which releases peptides with beneficial effects on the cholesterol metabolism after digestion. This study aimed to obtain peptides from Lupinus albus protein using an in vitro experimental model of human digestion and to evaluate the influence of peptides on the expression of genes involved in the intestinal and hepatic metabolism of cholesterol. Hydrolysates were obtained by using 2% (w:v) protein suspensions in NaCl 30 mM, pH 2.0. Pepsin was then added at enzyme substrate ratio of 1:1000 (E:S) and the digestion proceeded at 37 °C under agitation for 1h. Next, the pH was adjusted to 7.5 and added pancreatin at 1:1000 (E:S) and the reaction proceeded for 1h. To access the peptides, the hydrolysate was filtered through 3 kDa cutoff membrane, frozen and freeze-dried. The gene expression of NPC1L1, ABCA1 and ABCG1 was evaluated in intestinal Caco-2 cells treated with 0, 0.5, 1 and 5 mg/mL of peptides during 2, 6 and 12h. The hepatic LDLR and HMGCR mRNA expression was evaluated using HepG2 cells treated with 0, 0.3, 0.5, and 0.8 mg/mL of peptides during 24h. Total RNA was extracted from cells and mRNA expression was measured by Taqman qRT-PCR. NPC1L1 expression was increased after 6h with 5 mg/mL. ABCA1 mRNA level was induced after 2h exposure by all treatments. After 6h and 12h, ABCG1 expression was increased by the 5 mg/mL treatment. LDLR was not affected, whereas HMGCR showed a reduction for concentrations of 0.3 and 0.5 mg/mL. These results are suggestive that lupin bioactive peptides reduce the cholesterol synthesis in the liver and may influence the formation and maturation of HDL in intestinal cells through modulation of molecules involved in reverse cholesterol transport. These mechanisms maybe involved in the cholesterol-lowering effects associated with lupin protein ingestion.