Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human cardiovascular disease. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity, using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with RBP4 and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3 and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant, because HDL's APOE content associates with cardiovascular disease risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.