High density lipoproteins (HDL) facilitate reverse cholesterol transport (RCT), the process by which cholesterol effluxed from peripheral tissues is delivered via selective uptake to the liver for excretion. These processes are mediated by HDL's high-affinity receptor, scavenger receptor BI (SR-BI). SR-BI is an 82 kDa glycoprotein with two transmembrane domains (TMD), two short cytoplasmic N/C-terminal domains, and a large extracellular domain. Our laboratory solved the NMR structure of the C-terminal TMD and nearby extracellular domain. This peptide contains four conserved proline residues, an amino acid that confers structural flexibility. In our structure, P408 and P412 are in an extracellular alpha-helix, P438 lies at a helix-loop junction, and P459 introduces a kink into the TMD helix. We hypothesized that these proline residues support SR-BI-mediated cholesterol transport. To test this hypothesis, we mutated each proline to alanine and transiently expressed wild-type (WT), P408A-, P412A-, P438A-, or P459A-SR-BI in COS-7 cells. Total lysate and cell surface expression of SR-BI was examined by immunoblot analysis and flow cytometry, respectively. All mutants expressed on the cell surface, except P408A-SR-BI, which was absent from whole cell lysates. P412A- and P438A-SR-BI were significantly impaired in their ability to efflux cell-associated [ 3 H]cholesterol to HDL, as compared to WT-SR-BI. Likewise, cellular binding of [ 125 I]HDL and selective uptake of [ 3 H]cholesteryl oleyl ether were reduced with P412A- and P438A-SR-BI. We assessed SR-BI’s ability to modulate membrane cholesterol pools by cholesterol oxidase treatment. SR-BI enhances the accessibility of cholesterol to the exogenous enzyme, and a proportional increase in cholestenone is detected by thin layer chromatography. Cholestenone was increased with WT-SR-BI but not P412A- or P438A-SR-BI, suggesting defective membrane cholesterol distribution. Taken together, key proline residues in the near-C-TMD extracellular domain support efficient HDL binding, selective uptake, cholesterol efflux, and membrane cholesterol organization. These critical residues appear to support SR-BI in a conformation that allows for efficient RCT.