The essential requirement of the lymphotoxin beta receptor (LTβR) in the development and maintenance of peripheral lymphoid organs is well recognized. Evidence shows that LTβR is involved in various cellular processes; however, whether it plays a role in maintaining the cellular function of intestinal porcine enterocytes (IPEC-J2), specifically during porcine epidemic diarrhea virus (PEDV) infection, remains unknown. In this study, we generated LTβR null IPEC-J2 cells using CRISPR/Cas9 to examine the importance of LTβR in cell proliferation, apoptosis, and the response to PEDV infection. Our results showed that the lack of LTβR leads to significantly decreased cell proliferation, potentially due to S phase arrest in LTβR−/− IPEC-J2 cells. Label-free digital holographic microscopy was used to record the three-dimensional morphology of both cell types for up to 72 hours and revealed significantly increased numbers of LTβR−/− cells undergoing apoptosis. Furthermore, we found that PEDV-infected LTβR−/− null IPEC-J2 cells exhibited significant suppression of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) target genes (interleukin (IL)-6 and IL-8) and mucosal barrier integrity-related genes (vascular cell adhesion molecule 1 (VCAM1) and IL-22), which may explain why LTβR−/− cells are more susceptible to PEDV infection. Collectively, our data not only demonstrate the key role of LTβR in intestinal porcine enterocytes, but also provide data for the improved understanding of the cellular response to PEDV infection.