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Objective: HER2 overexpression is associated with aggressive phenotypes in breast cancer, including increased tumor proliferation, greater invasiveness, and reduced overall survival. The overall response rate to HER2-targeted therapies remains <30%. There is an urgent need for the identification of efficient markers to predict patients with a poor prognosis. This study was designed to investigate the correlation between EphB4 and EphrinB2 expression and the clinicopathological characteristics of HER2-positive breast cancer. Materials and methods: A total of 111 primary HER2-positive breast cancer patients were enrolled in this study (diagnosed since December 2005 to November 2010 from the Second Hospital of Dalian Medical University). The protein expression of EphB4 and EphrinB2 was examined by immunohistochemistry using paraffin-embedded tumor tissues. Results: There was a significant correlation between EphB4 and EphrinB2 expression (P=0.013, r=0.255). Kaplan-Meier analysis showed that the prognosis of patients with a high expression of both EphB4 and EphrinB2 was significantly worse than the prognosis of patients with either EphB4 or EphrinB2 expression and patients with negative expression (hazard ratio [HR] =1.935, P=0.0224). However, high expression of EphB4 or EphrinB2 alone was not an independent prognostic factor to predict worse overall survival. To summarize, HER2-positive breast cancer patients with overexpression of both EphB4 and EphrinB2 were associated with the worst prognosis. Conclusion: High expression of EphB4 and EphrinB2 correlated with poor overall survival, which can serve as an independent prognostic indicator in primary HER2-positive breast cancer patients. [ABSTRACT FROM AUTHOR]