Abstract: Protein disulfide-isomerase A3 (PDIA3), a member of the PDI family, plays a core role in regulating the formation, cleavage, and rearrangement of protein disulfide bonds. This enzyme is closely associated with the occurrence and progression of various physiological and pathological processes, including thrombosis, cancer, neurodegenerative diseases, and viral infections, making it a highly promising target for drug intervention. In this study, the gene encoding PDIA3 was introduced into an Escherichia coli expression system, and recombinant PDIA3 protein with biological activity was successfully expressed and purified. Based on the establishment of a high-throughput screening method for PDIA3 inhibitors, 110 small-molecule inhibitors of PDIA3 were identified from a natural product library comprising 4,080 compounds. Among these, Punicalagin, a major polyphenolic compound found in pomegranate peel, exhibited a relatively significant inhibitory effect, with a half-maximal inhibitory concentration (IC₅₀) of 2.3 μmol·L⁻¹. Molecular docking results suggested that Punicalagin may bind to the b' domain of PDIA3. This study provides candidate molecules and a theoretical basis for the development of PDIA3-targeted drugs.