We previously studied hypoxic tumor cells from hepatic metastases of colorectal cancer (CRC) and determined several potential prognostic factors, including expression of ephrin-A1 (EFNA1), which was highly induced by hypoxia. Here, we further evaluated the prognostic impact of EFNA1 expression. Samples from a total of 366 CRC patients from 11 institutes were analyzed by either microarray (n=220) or quantitative reverse-transcriptase polymerase chain reaction (n=146). EFNA1 was an independent prognostic factor for CRC (p<0.05). In vitro assays revealed that loss of EFNA1 following siRNA treatment was associated with reduced proliferative activity and decreased invasion and migration of CRC cell lines. EFNA1 expression is a useful marker for predicting high risk of relapse and cancer-related death in patients who have undergone curative resection for CRC.