Apolipoprotein A-IV, which is primarily synthesized in the intestine, is a multifunctional protein which is known to play a crucial role in the lipid metabolism. ApoA-IV is active in the reverse cholesterol transport pathway by activating lecithin-cholesterol acyltransferase and effluxing cellular cholesterol. Hence apoA-IV might prevent atherosclerosis. Moreover, apoA-IV, has many other physiological functions including acting as an antioxidant, an anti-inflammatory factor and an acute satiety factor. In addition, apoA-IV mediates glucose homeostasis by promoting insulin secretion from pancreatic islets. Wild type apoA-IV has a low lipid affinity, but the mutation of phenylalanine to alanine at position 334 generates apoA-IV (apoA-IV334) with higher affinity for lipid binding. In this study, recombinant human apoA-IVWT as well as the apoA-IV334 mutation were expressed in E.clean coli, a strain that contains a mutated lipopolysaccharide and does not contaminate the purified protein with endotoxin. The His tagged protein was purified by immobilized metal affinity chromatography followed by the removal of the His tag via an integral thrombin cleavage site. The cleaved His tag, thrombin and other contaminants were then separated in a second ion exchange purification step. ApoA-IVWT and apoA-IV334 were used to study the effects of the altered lipid affinity in vivo in an animal model of reverse cholesterol transport. Thus, 3[H]-cholesterol labelled macrophages as well as wild type and mutated apoA-IV334 were intraperitoneally injected into recipient mice. Levels of the 3[H]-tracer were measured in plasma, liver and faeces. In summary, results of this study showed that the altered lipid affinity of apoA-IV enhances the cholesterol efflux from macrophages and its faecal excretion, hence apoa-IV334 might be a more potent anti-atherogenic agent than apoA-IVWT.