Policosanol mixtures have been postulated as promising functional food ingredients to lower serum LDL cholesterol, and increase HDL cholesterol concentrations. We evaluated whether policosanol chain-length [tetracosanol (C24), hexacosanol (C26), octacosanol (C28), triacontanol (C30)] was relevant for its cholesterol lowering effects in heterozygous LDL-receptor deficient mice (LDLr +/-). In addition, effects of individual policosanols varying in chain-length and their respective long-chain fatty acids, and aldehydes on expression of genes involved in lipoprotein metabolism and apoA-I production, were evaluated in vitro. After a run-in period, LDLr +/- mice received experimental western-type diets without policosanols, or similar diets enriched with 30 mg/100 g individual policosanol, or a natural policosanol mixture (Lesstanol60). No significant effects on serum cholesterol concentrations, lipoprotein profiles, or hepatic and small-intestinal mRNA expression of lipoprotein metabolism related genes were found for any of the policosanol diets. In HepG2 and differentiated CaCo-2 cells, policosanols did not change de novo apoA-I protein production. In HepG2 cells, Lesstanol60 elevated gene expression of ABCA1 and HMG CoA synthase-1, however since effects were not observed in vivo, absorption of the responsible components seems to low. We conclude that individual policosanols as well as Lesstanol60 have no potential in reducing CHD risk through effects on serum lipoprotein concentrations.