We sought to evaluate the biological function of the receptor tyrosine kinase EphB4 in bladder cancer. All of the nine bladder cancer cell lines examined express EphB4 and the receptor could be phosphorylated following stimulation with its cognate ligand, EphrinB2. Out of the 15 fresh bladder cancer specimens examined, 14 expressed EphB4 with a mean sevenfold higher level of expression compared to adjacent normal urothelium. EphB4 expression was regulated by several mechanisms: EPHB4 gene locus was amplified in 27% tumor specimens and 33% cell lines studied; inhibition of EGFR signaling downregulated EphB4 levels; and forced expression of wild-type p53 reduced EphB4 expression. EphB4 knockdown using specific siRNA and antisense oligodeoxynucleotides molecules led to a profound inhibition in cell viability associated with apoptosis via activation of caspase-8 pathway and downregulation of antiapoptotic factor, bcl-xl. Furthermore, EphB4 knockdown significantly inhibited tumor cell migration and invasion. EphB4 knockdown in an in vivo murine tumor xenograft model led to a nearly 80% reduction in tumor volume associated with reduced tumor proliferation, increased apoptosis and reduced tumor microvasculature. EphB4 is thus a potential candidate as a predictor of disease outcome in bladder cancer and as target for novel therapy.