The effects of virus-like double-stranded RNA (dsRNA, PolyI:C) and DNA methyltransferase inhibitor (Aza-CdR) on CD4 gene expression were investigated in a porcine kidney cell line (PK15). We found that expression levels of TLR3 and IFNαwere significantly upregulated by PolyI:C, compared to the untreated PK15 cells, which shows that PolyI:C successfully mimics viral infection in PK15 cells. We also found that PolyI:C (10 μg/ml) and/or Aza-CdR (5 μM) significantly induces DNA demethylation of porcine CD4, promoting the binding of NF-κB to the CpG site on the CD4 promoter and activating expression of CD4. These data help clarify the regulatory mechanism of DNA methylation of the CD4 gene in non-immune cell response to virus replication. Further study is warranted to identify CD4 gene expression regulated by DNA methylation and live virus infection.