AbstractPorcine parvovirus (PPV) infection induces germ cell death, leading to reproductive disorders in first-pregnant sows. Porcine placental trophoblast cells (PTCs) are the major target of PPV, and we have previously found that PPV infection leads to the death of PTCs by a non-apoptotic process, which may be related to PPV pathogenicity. The Z-nucleic acid-binding protein 1 (ZBP1) is often activated after virus invasion and mediates subsequent cell death. Here, we found that PPV infection induced ZBP1-mediated necroptosis of porcine PTCs in the presence of the apoptosis inhibitor, AC-DEVD-CHO. ZBP1 expression was upregulated during PPV infection, and ZBP1 knockout or RNA interference significantly reduced its expression along with the PPV-induced necroptosis. We discovered that RIPK3 and MLKL, but not Caspase-8, were involved in downstream signaling of ZBP1 during PPV infection; the phosphorylation levels of RIPK3 and MLKL were enhanced, but Caspase-8 was not significantly cleaved. The knockout of RIPK3 and MLKL significantly reduced the PPV infection-induced necroptosis of porcine PTCs. RIPK3 knockout did not affect the PPV infection-induced upregulation of ZBP1 expression, but blocked the activation of MLKL. MLKL knockout did not affect the upregulation of ZBP1 expression and RIPK3 phosphorylation during PPV infection. UV-inactivated PPV induced significantly less necroptosis of porcine PTCs than non-irradiated PPV. A PPV strain with a mutation in the translation initiation codon was still able to induce necroptosis of PTCs through the ZBP1/RIPK3/MLKL pathway. These results provide new insights into the pathogenic mechanism of PPV infection and suggest that PPV infection of porcine PTCs induces necroptosis through the viral DNA-dependent ZBP1/RIPK3/MLKL pathway.