Pregnancy results in significant physiological changes which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well-documented with modest to no impact observed on the generic measure of high-density lipoprotein (HDL) cholesterol. However, no studies have taken a deeper look at the impact of pregnancy on the concentration and composition of HDL subspecies. In this prospective study, we collected plasma from 24 non-pregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR) we quantified 11 different lipoprotein size subspecies from plasma including 3 in the HDL class. We observed a profound increase in the number of larger HDL particles in pregnant women. These findings were confirmed by tracking phospholipid across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS) we identified 87 lipid-associated proteins across size speciated fractions. We report drastic shifts in multiple protein clusters across different HDL sized fractions in pregnant females compared to their non-pregnant controls. In summary, we have shown that pregnancy results in major alterations in HDL subspecies concentrations and compositions that would have otherwise been missed using traditional lipid measurements. These findings significantly elevate our understanding of how changes in lipoprotein metabolism due to pregnancy could impact the health of both the fetus and the mother.