Significance High-density lipoprotein (HDL), popularly known as “good cholesterol,” is an assembly of lipids and proteins that is protective against cardiovascular diseases. HDL begins to develop when two copies of the protein apolipoprotein A-I (APOA1) mediate the removal of excess lipids from peripheral cells and form a nanodisc. How the proteins adjust their structures as the nanodisc increases in diameter, mediates the conversion of cholesterol to cholesteryl esters, and becomes spherical is crucial to understanding HDL maturation and relevant pathologies. This study combines multimicrosecond atomistic molecular dynamics simulations, molecular modeling, and cross-linking experiments to reveal APOA1 structure in two nascent HDLs. The structures provide a mechanistic understanding for HDL maturation and revise previous models of nascent HDL.