Porcine epidemic diarrhea virus (PEDV), is the etiological agent of porcine epidemic diarrhea virus (PED), causes high mortality with severe vomiting, diarrhea, and dehydration in swine farms. In this study, the PEDV strain 85-7 could be proliferated effectively in MARC-145 cells, and caused a distinct inhibition of the expression of interferon-β (IFN-β, encoded by IFNB1), which suggested that a full understanding how this virus manipulates the host immune responses is critical in the fight against the spread of PEDV. We found that, the infection of PEDV strain 85-7 significantly downregulated HSP27 production in MARC-145 cells, and overexpression of HSP27 (encoded by HSPB1) decreased the virus titer by about 28-fold. Further study revealed that HSP27 could significantly activate the NF-κB phosphorylation, and thus increase the mRNA level of IFNB1 and downstream interferon-stimulated genes (ISGs) in MARC-145 cells. Indeed, treatment with IFN-β and the ISGs, including murine myxovirus resistance 1 and 2 (Mx1 and Mx2), showed direct anti-PEDV activity. Notably, the antiviral activity and transcription of the antiviral effectors induced by overexpression of HSP27 could be counteracted by the knockdown of IFNB1 via RNA interference, indicating that HSP27 was an upstream regulator of the intracellular antiviral effect against PEDV infection. This study is the first to link HSP27 to PEDV replication via the innate immunity response, which contributed to further clarify the mechanism of PEDV infection and the development of novel antiviral therapies.