H igh-density lipoprotein (HDL) particles play a pivotal role in reverse cholesterol transport (RCT) by facilitating cholesterol efflux from peripheral cells and delivering acquired lipid to the liver for elimination in the feces. 1 Obesity increases the risk of developing cardiovascular disease (CVD) 2 ; however, little is known about the impact of obesity on HDL function and RCT. Chronic inflammation is a classic hallmark of obesity 3 and CVD, 4 and it is plausible there is a common inflammatory-driven mechanism Background-Acute inflammation impairs reverse cholesterol transport (RCT) and reduces high-density lipoprotein (HDL) function in vivo. This study hypothesized that obesity-induced inflammation impedes RCT and alters HDL composition, and investigated if dietary replacement of saturated (SFA) for monounsaturated (MUFA) fatty acids modulates RCT. Methods and Results-Macrophage-to-feces RCT, HDL efflux capacity, and HDL proteomic profiling was determined in C57BL/6j mice following 24 weeks on SFA-or MUFA-enriched high-fat diets (HFDs) or low-fat diet. The impact of dietary SFA consumption and insulin resistance on HDL efflux function was also assessed in humans. Both HFDs increased plasma 3 H-cholesterol counts during RCT in vivo and ATP-binding cassette, subfamily A, member 1-independent efflux to plasma ex vivo, effects that were attributable to elevated HDL cholesterol. By contrast, ATP-binding cassette, subfamily A, member 1-dependent efflux was reduced after both HFDs, an effect that was also observed with insulin resistance and high SFA consumption in humans. SFA-HFD impaired liver-to-feces RCT, increased hepatic inflammation, and reduced ABC subfamily G member 5/8 and ABC subfamily B member 11 transporter expression in comparison with low-fat diet, whereas liver-to-feces RCT was preserved after MUFA-HFD. HDL particles were enriched with acute-phase proteins (serum amyloid A, haptoglobin, and hemopexin) and depleted of paraoxonase-1 after SFA-HFD in comparison with MUFA-HFD. Conclusions-Ex vivo efflux assays validated increased macrophage-to-plasma RCT in vivo after both HFDs but failed to capture differential modulation of hepatic cholesterol trafficking. By contrast, proteomics revealed the association of hepatic-derived inflammatory proteins on HDL after SFA-HFD in comparison with MUFA-HFD, which reflected differential hepatic cholesterol trafficking between groups. Acute-phase protein levels on HDL may serve as novel biomarkers of impaired liver-to-feces RCT in vivo. Correspondence to Fiona C. McGillicuddy, UCD Conway Institute, School of Medicine, University College Dublin, Dublin 4, Ireland. E-mail fiona.mcgillicuddy@ucd.ie © 2016 The Authors. Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited and is not used for commercial pur...