Cholesterol control is fundamental for prevention of cardiovascular disorders. In this work, the hypocholesterolemic activity of an aqueous Opuntia ficus-indica extract (AOE) was tested in triton-induced mice. The inhibitory activity on pancreatic lipase enzyme was evaluated in vitro by the same extract. Furthermore, polyphenol content of the extract was evaluated. Hypercholesterolemia was induced in three groups of mice by intraperitoneal administration of Triton WR-1339. After induction of hypercholesterolemia, the groups were treated with an AOE (500 mg/kg) and saline solution and the positive control group with orlistat, respectively. Cholesterol levels were measured 24 h later in peripheral blood. The levels of blood cholesterol after administration of AOE significantly decreased compared to negative control. The inhibitory activity of AOE on pancreatic lipase enzyme was evaluated at concentrations from 60 to 1000 μg/mL. The AOE inhibited the pancreatic lipase with an IC50 = 588.5 μg/mL. The AOE had a high content of polyphenolic compounds. These results show that AOE is able to prevent hypercholesterolemia by pancreatic lipase inhibition, in part due to its polyphenolic compounds.