Abstract: Protein disulfide-isomerase A3（PDIA3）, a member of the PDI family, primarily functions to regulate the formation, cleavage, and rearrangement of protein disulfide bonds, and is involved in a variety of physiological and pathological processes. In this study, a prokaryotic expression system was employed to prepare biologically active recombinant PDIA3 protein. Using an insulin reduction assay system, the enzyme activity determination method of PDIA3 protein was optimized by adjusting reaction conditions. Meanwhile, high-throughput screening of small-molecule inhibitors of PDIA3 protein was performed from a natural product library, and their inhibitory mechanisms were further explored. The results showed that the PDIA3 protein with a molecular weight of approximately 58 kDa was successfully expressed and purified via the Escherichia coli expression system. The optimized conditions for enzyme activity determination were as follows: temperature at 37℃, pH value of 7.0, and dithiothreitol（DTT）concentration of 0.2 mmol·L⁻¹. A total of 110 small molecules with inhibitory activity were initially screened out from 4,080 compounds, among which punicalagin exhibited a significant inhibitory effect with an IC₅₀ value of 2.3 μmol·L⁻¹. Molecular docking results indicated that punicalagin might bind to the b' domain of PDIA3 and inhibit its enzyme activity through hydrogen bonding and hydrophobic interactions. By determining PDIA3 protein activity and screening inhibitors, this study identified punicalagin, a small-molecule compound with strong inhibitory activity, from natural products, and preliminarily revealed its binding site. These findings provide candidate molecules and theoretical basis for the development of PDIA3-targeted drugs.