Abstract-Two common alleles exist at the haptoglobin (Hp) locus, and the Hp2 allele is associated with an increased incidence of cardiovascular disease, specifically in diabetes mellitus (DM). Oxidative stress is increased in Hp2 mice and humans with DM. Oxidative modification of the apolipoprotein A-I inhibits reverse cholesterol transport. We sought to test the hypothesis that reverse cholesterol transport is impaired in Hp2 DM mice and humans. In vitro, using serum from non-DM and DM individuals, we measured cholesterol efflux from 3 H-cholesterol-labeled macrophages. In vivo, we injected 3 H-cholesterol-loaded macrophages intraperitoneally into non-DM and DM mice with the Hp1-1 or Hp2-2 genotype and monitored 3 H-tracer levels in plasma, liver, and feces. In vitro, in DM individuals only, we observed significantly decreased cholesterol efflux from macrophages incubated with serum from Hp2-1 or Hp2-2 as compared with Hp1-1 individuals (PϽ0.01). The interaction between Hp type and DM was recapitulated using purified Hp and glycated Hb. In vivo, DM mice loaded with 3 H-cholesterol-labeled macrophages had a 40% reduction in 3 H-cholesterol in plasma, liver, and feces as compared with non-DM mice (PϽ0.01). The reduction in reverse cholesterol transport associated with DM was significantly greater in Hp2-2 mice as compared with Hp1-1 mice (54% versus 25% in plasma; 52% versus 27% in liver; 57% versus 32% in feces; PϽ0.03). reverse cholesterol transport is decreased in Hp2-2 DM. This may explain in part the increased atherosclerotic burden found in Hp2-2 DM individuals.