Protein disulfide isomerase 3 (PDIA3) is a chaperone protein that modulates the folding of newly synthesized glycoproteins, has isomerase and redox activity, and has been implicated in the pathogenesis of many diseases. However, the role of PDIA3 in pregnancy-associated diseases remains largely unknown. Our present study reveals a key role for PDIA3 in the biology of placental trophoblasts from women with preeclampsia (PE). Immunohistochemistry and Western blot analysis revealed that PDIA3 expression was decreased in villous trophoblasts from women with PE compared to normotensive pregnancies. Further, using a Cell Counting Kit-8 assay, flow cytometry, and 5-ethynyl-2’-deoxyuridine (EdU) staining, we found that siRNA-mediated PDIA3 knockdown significantly promoted apoptosis and inhibited proliferation in the HTR8/SVneo cell line, while overexpression of PDIA3 reversed these effects. Furthermore, RNA sequencing and Western blot analysis demonstrated that knockdown of PDIA3 inhibited MDM2 protein expression in HTR8 cells, concurrent with marked elevation of p53 and p21 expression. Conversely, overexpression of PDIA3 had the opposite effects. Immunohistochemistry and Western blot further revealed that MDM2 protein expression was downregulated and p21 was increased in trophoblasts of women with PE compared to women with normotensive pregnancies. Our findings indicate that PDIA3 expression is decreased in the trophoblasts of women with PE, and decreased PDIA3 induces trophoblast apoptosis and represses trophoblast proliferation through regulating the MDM2/p53/p21 pathway.