Policosanol has been reported to improve blood pressure, lipid profile, and HDL functionality via inhibition of cholesteryl ester transfer protein (CETP) both in vitro and in vivo in zebrafish and human models. However, there are limited reports and randomized, double-blinded trials on policosanol that could advocate the blood pressure-lowering effect in prehypertensive participants. Therefore, we performed in vitro, in vivo, and ex vivo experiments to provide more substantial and concrete data on the blood pressure-lowering effect of policosanol. Consumption of policosanol for 8 weeks enhanced plasma antioxidant activity. In the policosanol group, plasma total cholesterol (TC) and triglyceride (TG) levels were reduced up to 20% and 14%, respectively, and HDL-C level was elevated up to 1.3-fold compared to that at week 0. TG/HDL-C and cholesteryl ester transfer protein (CETP) activities were reduced up to 36% and 20%, respectively. Uptake of oxidized LDL in macrophages was reduced as oxidized species levels were reduced, and HDL2-associated paraoxonase activities were enhanced by 60% compared to those at week 0. Encapsulation of policosanol into reconstituted HDL (PCO-rHDL) enhanced cholesterol efflux activity and insulin secretion capacity. In conclusion, consumption of policosanol for 8 weeks in healthy female subjects resulted in lowered blood pressure and CETP activity via elevation of HDL/apoA-I contents and enhancement of HDL functionalities, including cholesterol efflux and insulin secretion. These functional enhancements of HDL can contribute to the prevention of aging-related diseases, hypertension, and stroke.