The mechanisms by which the cholesterol-conjugated siRNA (Chol-siRNA) molecules are transferred from reconstituted high density lipoprotein (rHDL) into targeted tumor cells have been expounded in the present study.By using fluorescent-tagged molecules of apolipoprotein A-I (apoA-I) and Chol-siRNA, the observations from FACS and confocal microscopic analyses confirmed the formation of Chol-siRNA-loaded rHDL nanoparticles (rHDL/Chol-siRNA complexes) and the attachment of apoA-I on the surface of Chol-siRNA-loaded lipoplexes (Lipos/Chol-siRNA complexes).Well formulated rHDL/Chol-siRNA complexes demonstrated proper nanosize, quasi-spherical shapes and increased nuclease protection over naked Chol-siRNA.Cellular distribution and internalization pathway of Chol-siRNA in rHDL/Chol-siRNA complexes was confirmed in vitro; the fate of Chol-siRNA cargo from that of rHDL/Chol-siRNA complexes components was distinguished in SR-BI high-expressing tumor cells, such as human hepatocellular carcinoma cell line HepG2 and human breast cancer cell line MCF-7.The results revealed that rHDL provided an extraordinary and highly effective approach for cross-membrane transfer of Chol-siRNA into the cytoplasm via the scavenger receptor BI (SR-BI)-mediated non-endocytotic mechanism.In addition, in vivo tumor-selective targeting behavior and biodistribution of Cy5 labeled Chol-siRNA-loaded rHDL nanoparticles (rHDL/Cy5-Chol-siRNA complexes) were studied using non-invasive near-infrared fluorescence imaging system in nude mice simultaneously inoculated with MCF-7 cells (SR-BI+) and HT1080 cells (SR-BI-), coupled with measurements of the Chol-siRNA positive cells percentage in each tumors.The complexes exhibited long circulation time, SR-BI positive tumor-selective targeting ability.Moreover, rHDL/Chol-siRNA complexes also exhibited less hemolysis than PEI commonly used as gene carrier, as well as no local venous irritation and hypersensitivity.Therefore, rHDL is proposed as a considerable potential and versatile candidate, mediating Chol-siRNA target-specific cytosolic delivery for tumor gene silencing therapy.