Elevated plasma triglycerides (TGs) are causally associated with coronary artery disease and acute pancreatitis. Apolipoprotein A-V (apoA-V) circulates on TG-rich lipoproteins (TRLs) and promotes lipoprotein lipase (LPL) enzymatic activity, thereby decreasing plasma TG levels. While apoA-V’s association with plasma TGs in humans is clear, the mechanisms by which apoA-V influences TG metabolism are not fully understood. I sought to elucidate part of apoA-V’s role in TG metabolism using naturally occurring variants to interrogate protein structure-function. I used a novel experimentally-derived secondary structure of human apoA-V to identify a C-terminal hydrophobic face, and genomic data to identify a very rare C-terminal truncation variant predicted to eliminate this region. ApoA-V Q252X was associated with elevated plasma TG levels in humans and this phenotype was recapitulated in apoa5 knockout (KO) mice expressing the variant. Part of this loss of function (LoF) phenotype was explained by decreased mRNA expression. Functionally, recombinant apoA-V Q252X protein was more readily soluble in aqueous solutions, more exchangeable with lipoproteins than wildtype (WT) apoA-V, and decreased plasma TG in vivo. I also used the secondary structure and genomic data to identify several putative gain of function (GoF) variants in apoA-V. I developed a humanized in vitro LPL TG hydrolysis assay which captures apoA-V activity and includes the LPL chaperone glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and a structured TRL substrate. I also optimized in vitro methods for the study of intracellular apoA-V in apolipoprotein B (apoB) and TG secretion and in vivo methods that capture apoA-V’s effect on TG metabolism following lipid challenge. My studies demonstrate that deletion of apoA-V’s C-terminus leads to reduced apoA-V bioavailability and higher TG levels. Removal of the C-terminus markedly reduces apoA-V aggregation while maintaining apoA-V lipoprotein binding and intravascular activity. This variant-driven structure-function assessment of apoA-V, a key player in TG metabolism, has added to our understanding of the biology of this protein and how we can leverage its activity in the development of novel TG-lowering therapeutics.