Abstract Plasma high-density lipoprotein (HDL) protect endothelial cells against apoptosis induced by oxidatively modified low-density lipoprotein (oxLDL). The specific component(s) of HDL implicated in such cytoprotection remain(s) to be identified. Human microvascular endothelial cells (HMEC-1) were incubated with mildly oxLDL in the presence or absence of each of five physicochemically-distinct HDL subpopulations fractionated from normolipidemic human plasma (n=7) by isopycnic density gradient ultracentrifugation. All HDL subfractions protected HMEC-1 against oxLDL-induced primary apoptosis as revealed by nucleic acid staining, annexin V binding, quantitative DNA fragmentation, inhibition of caspase-3 activity, and reduction of cytoplasmic release of cytochrome c and apoptosis-inducing factor. Small, dense HDL3c displayed twofold superior intrinsic cytoprotective activity (as determined by mitochondrial dehydrogenase activity) relative to large, light HDL2b on a per particle basis (p<0.05). Equally, all HDL subfractions attenuated intracellular generation of reactive oxygen species (ROS); such antioxidative activity diminished from HDL3c to HDL2b. The HDL protein moiety, in which apolipoprotein A-I (apoA-I) predominated, accounted for approximately 70% of HDL antiapoptotic activity. Furthermore, HDL reconstituted with apoA-I, cholesterol and phospholipid potently protected HMEC-1 from apoptosis. By contrast, modification of the content of sphingosine-1-phosphate in HDL did not significantly alter cytoprotection. We conclude that small, dense, lipid-poor HDL3 potently protect endothelial cells from primary apoptosis and intracellular ROS generation induced by mildly oxLDL, and that apoA-I is pivotal to such protection.