Obesity is a chronic disease that has been causing serious problems all over the world. However, there is a lack of available therapeutic approaches to treat obesity. The FDA-approved drug orlistat has severe side effects, such as abdominal pain, flatulence and oily stool. As the therapeutic target of orlistat is pancreatic lipase, there is an urgent need for discovery of new pancreatic lipase inhibitors from natural sources that have reduced side effects compared with orlistat. In this study, ultrafiltration in combination with molecular simulation and spectroscopy was reported as an effective approach for identifying new pancreatic lipase inhibitors from anthocyanin-rich berry sources. Using this approach, four monomeric anthocyanins cyanidin-3-O-arabinoside (C3A), cyanidin-3-O-galactoside (C3Ga), peonidin-3-O-arabinoside (Pn3A) and peonidin-3-O-galactoside (Pn3Ga) from cranberries were discovered as potent pancreatic lipase inhibitors. These four cranberry anthocyanins were shown to form hydrophobic interactions and hydrogen bonds with pocket amino acid residues in molecular docking and molecular dynamics simulations. C3A showed greater impact on secondary structures of the enzyme and showed higher binding capacity with the enzyme compared with C3Ga, Pn3A and Pn3Ga as observed by CD and fluorescence spectroscopy. The structure-activity relationships were then investigated and summarized as both the structures of the B ring and glycosyl group were related to the inhibitory activities of anthocyanins. In short, our results suggested that cranberry anthocyanins could be developed as food supplements to facilitate the prevention and treatment of obesity.