To isolate novel diagnostic markers and drug targets for pancreatic ductal adenocarcinoma (PDAC), we previously performed expression profile analysis of PDAC cells using a genome-wide cDNA microarray combined with laser microdissection. Among dozens of up-regulated genes identified in PDAC cells, we herein focused on one tyrosine kinase receptor, Eph receptor A4 (EphA4), as a molecular target for PDAC therapy. Immunohistochemical analysis validated EphA4 overexpression in approximately half of the PDAC tissues. To investigate its biological function in PDAC cells, we knocked down EphA4 expression by siRNA, which drastically attenuated PDAC cell viability. In concordance with the siRNA experiment, PDAC-derivative cells that were designed to constitutively express exogenous EphA4 showed a more rapid growth rate than cells transfected with mock vector, suggesting a growth-promoting effect of EphA4 on PDAC cells. Furthermore, the expression analysis for ephrin ligand family members indicated the coexistence of ephrinA3 ligand in PDAC cells with EphA4 receptor, and knockdown of ephrinA3 by siRNA also attenuated PDAC cell viability. These results suggest that the EphA4-ephrinA3 pathway is likely to be a promising molecular target for pancreatic cancer therapy.