Scavenger receptor, class B, type I (SR-BI) mediates selective uptake of cholesterol from high density lipoprotein (HDL) particles, a poorly understood process that is distinct from endocytic uptake of lipoproteins, such as low density lipoprotein (LDL). We set out to find small molecules that modulate SR-BI function and could be used to characterize the mechanisms involved in HDL uptake. Using a cell-based DiI-HDL uptake assay, we performed a high-throughput screen (HTS) of the National Institutes of Health Molecular Libraries Probe Centers Network (NIH MLPCN) compound library. Of 319,533 compounds, 3,046 compounds (0.96%) were classified as inhibitors of DiI-HDL uptake. A bis-amide (CID 650573) was identified in the primary HTS as an inhibitor. It had potent activity upon retesting in the primary assay, a low hit rate in other MLPCN assays, and possessed structural properties suitable for analog synthesis. Structure activity relationship (SAR) studies were performed to improve potency and to minimize deleterious properties. These efforts generated a probe (CID 53393835/ML279), a low nanomolar inhibitor with improved potency and decreased metabolic liability. ML279 was tested for efflux inhibition, modulation of HDL binding to SR-BI, and inhibition of endocytosis. ML279 functions by inhibiting both SR-BI-mediated uptake and efflux of free cholesterol to HDL particles. As a tool compound, ML279 is superior to existing small-molecule inhibitors of SR-BI (e.g., BLT-1 and ITX-5061) and will be useful in elucidating how SR-BI mediates lipid transport. In addition, it could clarify the role of SR-BI in a number of biological processes where it plays a crucial role.