CD36, a member of the class B scavenger receptor, is a high-affinity receptor for oxidatively modified low-density lipoprotein (oxLDL). Extensive evidence points to a significant role of CD36 in atherosclerosis and suggests that CD36 could be a potential target for treatment of atherosclerosis. Here, the extracellular domain of human CD36 (Gly(30)-Asn(439)) was expressed in Escherichia coli as His(6)-tagged soluble CD36 (sCD36), which could bind oxLDL specifically and effectively inhibit the uptake of oxLDL by murine macrophage RAW 264.7 cells. An enzyme-linked immunosorbent assay (ELISA)-like high-throughput screening (HTS) assay was developed for the discovery of CD36 antagonists, based on the competition of sCD36 binding to immobilized oxLDL and detection with a monoclonal antibody against His-tag. This assay was suitable for HTS in a 96-well format and was robust and reliable according to the evaluation parameter Z' value of 0.82. The developed HTS assay was applied to both pure chemical compounds and microbial secondary metabolite crude extracts to identify CD36 antagonists. Three active compounds-sodium danshensu (DSS), rosmarinic acid (RA), and salvianolic acid B (SAB)-were shown to be antagonistic to sCD36-oxLDL binding and further validated by their inhibition of oxLDL uptake in RAW 264.7 cells. These results suggest that the ELISA-like assay represents a promising screening for identifying bioactive molecules targeting atherosclerosis at the level of CD36-ligand binding.