Description: BACKGROUND Although SDF-1/CXCR4 pathway is a potential mechanism of tumor proliferation and progression, the mechanism of controlling CXCR4 expression is not fully understood. This study was to confirm that miR-494-3p might be a potentially post-transcriptional regulator of CXCR4 and over-expression of miR-494 might suppress prostate cancer progression and metastasis. MATERIALS AND METHODS We firstly postulated the post-transcriptional regulation of CXCR4 by miR-494-3p through bioinformatics analysis, and then it was demonstrated that miR-494-3p could regulate the CXCR4 mRNA post-transcriptionally by binding to the predicted site by dual reporter gene assays. The biological effect of miR-494-3p on prostate cancer cells proliferation, apoptosis, migration, and invasion was measured by MTT, TUNEL, flow cytometry, migration, and invasion assays. RESULTS It was shown that the mRNA and protein expression levels of CXCR4 were significantly up-regulated in PC-3 and DU145, whereas barely detected in LNCaP and RWPE-1. However, the CXCR4 protein levels were inversely related to the mature miR-494-3p expression levels in RWPE-1 and prostate cancer cells. The constitutive over-expression of miR-494-3p could down-regulate the protein level of CXCR4 in PC-3 and DU145. MiR-494-3p also could bind to the seed sequences in the 3′-UTR of the CXCR4 gene. Artificial over-expression of miR-494-3p could inhibit the growth, promote the apoptosis, and inhibit the migration and invasion of PC-3 and DU145 cells in vivo. CONCLUSIONS Our results suggested that miR-494-3p might play crucial role in prostate cancer by post-transcriptional regulation to CXCR4 mRNA. MiR-494-3p/CXCR4 pathway may be a potential therapeutic target to prevent prostate cancer progression and metastasis. Prostate © 2014 Wiley Periodicals, Inc.