Porcine epidemic diarrhea virus (PEDV) is a coronavirus that induces persistent diarrhoea in swine, resulting in severe economic losses in swine-producing countries. Insights into the interplay between PEDV infection and the innate immune system are necessary for understanding the associated mechanism of pathogenesis. The transcription factor NF-κB plays an important role in regulating host immune responses. Here, we elucidated for the first time to our knowledge the potential mechanism of PEDV-mediated NF-κB activation in porcine small intestinal epithelial cells (IECs). During PEDV infection, NF-κB p65 was found to translocate from the cytoplasm to the nucleus, and PEDV-dependent NF-κB activity was associated with viral dose and active replication. Using small interfering RNAs to screen different mRNA components of the Toll-like receptor (TLR) or RIG-I-like receptor signalling pathways, we demonstrated that TLR2, TLR3 and TLR9 contribute to NF-κB activation in response to PEDV infection, but not RIG-I. By screening PEDV structural proteins for their ability to induce NF-κB activities, we found that PEDV nucleocapsid protein (N) could activate NF-κB and that the central region of N was essential for NF-κB activation. Furthermore, TLR2 was involved in PEDV N-induced NF-κB activation in IECs. Collectively, these findings provide new avenues of investigation into the molecular mechanisms of NF-κB activation induced by PEDV infection.