EphB4 is a receptor tyrosine kinase that is over-expressed in 66% of prostate cancers (PCa) and can regulate cell migration and invasion. It is recognized as a potentially important therapeutic target. The pathways by which EphB4 promotes tumour progression are poorly understood. To examine the molecular networks that EphB4 influences in PCa, we transiently knocked down EphB4 or over-expressed EphB4 in LNCaP cells, followed by cDNA microarray/bioinformatic analysis. Quantitative real-time PCR and western immunoblotting of selected target genes/proteins validated their differential, EphB4-mediated expression. Microarray analysis identified 260 up-regulated genes and 300 down-regulated genes when EphB4 was knocked down (by 70 %) in LNCaP PCa cells. Gene ontology analysis showed the process of cell adhesion as being most significantly influenced. Several integrins appeared to be deregulated, but Integrin β8 (ITGB8) was the top hit with a 29-fold down-regulation in EphB4 siRNA treated cells, compared to control cells (treated with a scrambled, control siRNA). Over-expression of EphB4 led to a simultaneous increase in ITGB8 expression. Integrins play an essential role in the communication between cells and the extracellular matrix, influencing adhesion, migration and invasion of cancer cells. Whilst several members of the integrin family have been a focus in PCa, nothing is known about the role of ITGB8 in this disease. Analysis using the Oncomine clinical cohort database revealed that ITGB8 and EphB4 are both highly expressed in prostatic intraepithelial neoplasms (PIN) with decreasing expression in prostate carcinomas and basal expression in metastases, suggesting roles in the early stages of PCa progression. In conclusion, we have discovered that EphB4 regulates ITGB8 expression, that they are concomitantly expressed in PCa and that both are highly expressed in PIN. This suggests that EphB4 and ITGB8 could be involved in the onset of PCa and targeting these two proteins synergistically may impact on PCa progression.