HDL proteome dynamics may determine HDL cardioprotective functions. We aimed to characterize proteome and lipid profiles in small, medium and large (S/M/L) HDL fractions and analyze their functions. We characterized mouse HDL fractions by their high phospholipid (PL) content and collected S/M/L-HDLsubfractions by using fast protein liquid chromatography. The pooled S/L/M-HDL elution was subjected to mass spectrometry (MS) analysis using a Qstar XL-MS system, performing HDL subpopulation proteomic analysis. Fifty-one HDL proteins (39 in S-HDL, 27 in M-HDL and 29 in L-HDL) were identified and grouped into 4 functional categories (5 in lipid metabolism, 24 in immune response, 7 in coagulation, and 14 others). There were 16, 3 and 7 proteins present in only the S-HDL, M-HDL and L-HDL subfractions, respectively, and 11 proteins overlapped in all S/M/L-HDL subfractions. The dynamic changes in relative HDL protein levels were then characterized based on their functional groups by biological and bioinformatical methods.