Seneca Valley Virus (SVV) or commonly known as Senecavirus A (SVA), is one of picornavirus that is associated with vesicular disease and neonatal mortality in swine herds. However, the pathogenesis of SVV remains largely elusive. Our previous study found that SVV replicates extremely faster in porcine IBRS-2 cells than that in porcine PK-15 cells. However, the underlying mechanism remains unknown. In this study, we comprehensively compared the expression features between IBRS-2 cells and PK-15 cells in response to SVV infection by an unbiased high-throughput quantitative proteomic analysis. We found that the innate immune response-realted pathways were efficiently activated in PK-15 cells but not in IBRS-2 cells during SVV infection. A large amount of interferon-stimulated genes (ISGs) were induced in PK-15 cells. In contrast, no ISGs were induced in IBRS-2 cells after SVV infection. This different expression features in the two cell lines was verified by qPCR analysis. Besides, we determined similar results in the two cell lines during another porcine picornavirus foot-and-mouth disease virus (FMDV) infection. Further study demonstrated that the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway was functioning properly in both IBRS-2 and PK-15 cells. A systematic screening study revealed that the aberrant signal transduction from TBK1 to IRF3 in the RIG-I-like receptor signaling pathway in IBRS-2 cells was the fundamental cause of the different innate immune response manifestation and different viral replication rate in the two cell lines. Together, our findings determined the different feature of IBRS-2 and PK-15 cell lines which will provide useful guidance for choosing right cell line in porcine picornaviruses-mediated host immune signaling research and could be easily extended to other porcine viruses.