Farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, plays an important role in the regulation of cholesterol and more specifically high-density lipoprotein (HDL) homeostasis. Activation of FXR is reported to lead to both pro- and anti-atherosclerotic effects. In the present study we analyzed the impact of different FXR agonists on cholesterol homeostasis, plasma lipoprotein profiles, and transhepatic cholesterol efflux in C57BL/6J mice and cynomolgus monkeys and atherosclerosis development in cholesteryl ester transfer protein transgenic (CETPtg) low-density lipoprotein receptor (LDLR) (-/-) mice. In C57BL/6J mice on a high-fat diet the synthetic FXR agonists isopropyl 3-(3,4-difluorobenzoyl)-1,1-dimethyl-1,2,3,6-tetrahydroazepino[4,5-b]indole-5-carboxylate (FXR-450) and 4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl]benzoic acid (PX20606) demonstrated potent plasma cholesterol-lowering activity that affected all lipoprotein species, whereas 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) and 6-ethyl chenodeoxycholic acid (6-ECDCA) showed only limited effects. In FXR wild-type mice, but not FXR(-/-) mice, the more efficacious FXR agonists increased fecal cholesterol excretion and reduced intestinal cholesterol (re)uptake. In CETPtg-LDLR(-/-) mice PX20606 potently lowered total cholesterol and, despite the observed HDL cholesterol (HDLc) reduction, caused a highly significant decrease in atherosclerotic plaque size. In normolipidemic cynomolgus monkeys PX20606 and 6-ECDCA both reduced total cholesterol, and PX20606 specifically lowered HDL(2c) but not HDL(3c) or apolipoprotein A1. That pharmacological FXR activation specifically affects this cholesterol-rich HDL(2) subclass is a new and highly interesting finding and sheds new light on FXR-dependent HDLc lowering, which has been perceived as a major limitation for the clinical development of FXR agonists.