BACKGROUND/AIM: We sought to identify the mechanisms of perineural invasion in pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: We utilized in vitro cancer cell-nerve co-culture models comprising human PDAC cell lines (MIA Paca2 and PANC-1) and a dorsal root ganglion (DRG) isolated from neonatal mice. We compared gene expression profiles between cell lines with/without DRG conditioned medium (DRG-CM) using RNA-sequencing (RNA-seq). RESULTS: Migration, invasion, and neurotropism were significantly enhanced in MIA Paca2 but not in PANC-1 cells co-cultured with DRGs. Among 285 genes which showed significant differences in expression levels between cell lines in RNA-seq, we focused on Ephrin receptor A4 (EPHA4), which was upregulated in MIA Paca2 cells treated with DRG-CM. The abilities of migration, invasion, and neurotropism enhanced by DRG co-culture were abolished when EPHA4 was knocked down by siRNA in MIA Paca2 cells. CONCLUSION: EPHA4 can be a potential target gene to regulate perineural invasion in PDAC cells.