Previous studies demonstrated that peptides produced by the hydrolysis of olive seed proteins using Alcalase enzyme showed in vitro multifunctional lipid-lowering capability. This work presents a deeper insight in the hypolipidemic effect of olive seed peptides. The capability of olive seed peptides to inhibit endogenous cholesterol biosynthesis through the inhibition of HMG-CoA reductase enzyme was evaluated observing a 38 ± 7% of inhibition. Two in vivo assays using different peptides concentrations (200 and 400 mg/kg/day) were designed to evaluate the hypolipidemic effect of olive seed peptides in male and female mice. A low concentration of hydrolysate reduced total cholesterol in male mice in a 20% after 11 weeks compared to the mice feeding the hypercholesterolemic diet. A higher hydrolysate concentration showed a greater reduction in total cholesterol (25%). The analysis of the olive seed hydrolysate by RP-HPLC-MS/MS enabled the identification of peptides that could be responsible for this hypolipidemic effect.