Abstract Body: Dyslipidemia is a risk factor for metabolic diseases, emphasizing the importance of regulating lipoprotein metabolism. Levels of lipoproteins are regulated, in part, by extracellular lipases including lipoprotein lipase (LPL) and endothelial lipase (EL). Angiopoietin-like-3 (ANGPTL3) regulates both EL and LPL activity, inhibiting EL on its own and LPL when ANGPTL3 forms a complex with angiopoietin-like-8 (ANGPTL8). ANGPTL3 and ANGPTL3-8 complexes are both considered targets for the treatment of dyslipidemia. Understanding the residues of ANGPTL3 that contribute to each of its functions could enable the development of more effective therapies. The goal of our study was to identify residues in ANGPTL3 that contribute to complex formation, EL inhibition, and LPL inhibition. We generated a panel of mutations that spanned the N-terminal domain of ANGPTL3, and the ability of each mutant to inhibit EL and LPL was assessed using lipase activity assays. The oligomerization state of wild-type and mutant ANGPTL3 and wild-type and mutant ANGPTL3-ANGPTL8 complexes was determined using mass photometry and blue native gel electrophoresis. We found that mutations in the SE1 domain and surrounding residues disrupted the ability of ANGPTL3 to inhibit EL or LPL. Several leucine residues throughout the N-terminal region were also essential for lipase inhibition. Our oligomeric studies found that wild-type ANGPTL3 primarily forms a homotrimer and that trimer formation is necessary for the inhibition of EL. Mutations that disrupted trimer formation, which included the leucine mutations we identified in our inhibition studies, had no capacity to inhibit EL. Blue native electrophoresis with ANGPTL3-ANGPTL8 strongly suggests that these complexes form as trimers in a two ANGPTL3 to one ANGPTL8 ratio. Our results also show that some residues of ANGPTL3 are critical for inhibiting both EL and LPL, but some are specifically important only for EL or LPL inhibition. Identification of these residues allows us to dissect distinct pathways by which ANGPTL3 regulates lipoprotein metabolism.