microRNAs (miRNAs/miRs) are a class of single-stranded non-coding RNA molecules of 19-24 nucleotides in length. Via specific mRNA complementary paring of target genes, miRNAs are able to regulate the expression of mRNA levels or inhibit protein translation following transcription. miRNAs can act as oncogenes or tumor suppressors. We have previously reported that miR-26b was expressed at a lower level in PCa cells compared to normal prostate cells and it inhibited autophagy. Here, we further revealed the role of miR-26b in prostate cancer cells. We found that over-expression of miR-26b suppressed prostate cancer cell proliferation, invasion and migration in vitro and inhibited the growth of prostate xenograft tumor in vivo. We have processed a gene expression microarray assay to investigate the concrete mechanism of miR-26b inhibition on prostate cancer cells proliferation and migration. We found that miR-26b significantly up-regulated 57 genes expression level, and simultaneously down-regulated 55 genes expression (fold change >2; P < 0.05) in PC-3. The differential genes were most associated with the regulation process of cell proliferation, apoptotic process, protein phosphorylation and ubiquitination respectively, and enriched in multiple pathways including TNF signaling pathway and TGF-β signaling pathway. Among these filtered genes, CEACAM6 was significantly down regulated by miR-26b with a 2.17-fold. We identified a putative miR-26b binding site on 3′UTR region of CEACAM6 and validated that miR-26b bound to the 3′UTR region of CEACAM6 mRNA, suggesting that CEACAM6 is a direct target of miR-26b. Our results suggest that miR-26b suppresses cell proliferation by targeting CEACAM6 in PCa cells and miR-26b may be a candidate tumor-suppressor in prostate cancer.