Background: Increased expression of erythropoietin-producing human hepatoma (EPHB4) leads to enhanced cell migration, growth and adhesion in tumor cells. However, little is known regarding the effects of EPHB4 in gastric cancer. The present study aimed to examine the clinical relevance of EPHB4 and its association with the prognosis of gastric cancer. Materials and Methods: EPHB4 transcript expression in 324 gastric cancer samples with paired adjacent normal gastric tissues was determined using quantitative polymerase chain reaction and the results were statistically analyzed against patient clinicopathological data. AGS and HGC27 cell lines were transfected with EPHB4 siRNA and the effects examined by functional analysis. Results: EPHB4 mRNA levels in gastric cancer tissues were significantly elevated when compared to non-cancerous tissues (p=0.0110). Tissue samples from male patients exhibited lower expression than those from female patients (p=0.0110). Non-cardiac gastric tumors (fundus, corpus and pylorus) expressed a higher number of EPHB4 transcripts in comparison to cardiac gastric tumors (p<0.001). Increased expression of EPHB4 was significantly associated with poorer overall (p=0.0051) and progression-free (p=0.0262) survival. EPHB4 knockdown appeared to reduce post-wound migration of AGS cells (p=0.0057) and increase migration of HGC27 cells (p=0.0337). EPHB4 knockdown significantly increased adhesive ability in HGC27 (p<0.0001). Conclusion: The expression of EPHB4 was increased in gastric cancer and increased EPHB4 expression was correlated with poor survival. Knockdown of EPHB4 promoted adhesion and exerted diverse effects on migration of gastric cancer cells. Further investigations may highlight its predictive and therapeutic potential in gastric cancer.