Lipoprotein lipase (LPL) carries out the lipolytic processing of triglyceride-rich lipoproteins (TRL) along the luminal surface of capillaries. LPL activity is regulated by the angiopoietin-like proteins (ANGPTL3, ANGPTL4, ANGPTL8), which control the delivery of TRL-derived lipid nutrients to tissues in a temporal and spatial fashion. This regulation of LPL mediates the partitioning of lipid delivery to adipose tissue and striated muscle according to nutritional status. A complex between ANGPTL3 and ANGPTL8 (ANGPTL3/8) inhibits LPL activity in oxidative tissues, but its mode of action has remained unknown. Here, we used biophysical techniques to define how ANGPTL3/8 and ANGPTL3 interact with LPL and how they drive LPL inactivation. We demonstrate, by mass photometry, that ANGPTL3/8 is a heterotrimer with a 2:1 ANGPTL3:ANGPTL8 stoichiometry and that ANGPTL3 is a homotrimer. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies revealed that ANGPTL3/8 and ANGPTL3 use the proximal portion of their N-terminal α-helices to interact with sequences surrounding the catalytic pocket in LPL. That binding event triggers unfolding of LPL's