Overexpression of elongation factor-1α (EF-1α) has been reported to contribute to the development and progression of various cancers. However, its role in prostate cancer (PCa) still remains poorly understood. In the present study, we investigate the influence of EF-1α in Du145, a high-grade metastatic PCa cell line, and demonstrate that EF-1α plays an essential role in cellular properties associated with tumor progression, namely cell proliferation, invasion, and migration. In this study, EF-1α expression in human PCa cell line Du145 was reduced by RNA interference (RNAi) technology, and the proliferation, invasion, and migration of EF-1α-reduced Du145 cells were examined. We also detected an EF-1α expression pattern in 20 pairs of primary PCa samples and their corresponding normal tissues. Expression of EF-1α was detectable in four PCa cell lines (22RV1, LnCap, Du145, and PC3), indicating its possible role in pathogenesis of PCa. RNAi-mediated knockdown of EF-1α expression in Du145 cells, which expressed the highest level of EF-1α among four PCa cell lines, led to a decrease in proliferation. Similarly, suppression of EF-1α inhibited Du145 cell migration and invasion through a basement membrane substitute. Furthermore, we found that the normal prostate tissues showed a relatively low level of EF-1α expression, whereas PCa tissues demonstrated significantly higher expression levels of EF-1α (P < 0.001). Taken together, these findings support the hypothesis that EF-1α affects multiple processes involved in tumor progression, and identify EF-1α as a potential therapeutic target.