MicroRNAs (miRNA) have been globally profiled in cancers but there tends to be poor agreement between studies including in the same cancers. In addition, few putative miRNA targets have been validated. To overcome the lack of reproducibility, we profiled miRNAs by next generation sequencing and locked nucleic acid miRNA microarrays and verified concordant changes by quantitative RT-PCR. Notably, miR-125b and the miR-99 family members miR-99a, -99b, and -100 were downregulated in all assays in advanced prostate cancer cell lines relative to the parental cell lines from which they were derived. All four miRNAs were also downregulated in human prostate tumor tissue compared with normal prostate. Transfection of miR-99a, -99b, or -100 inhibited the growth of prostate cancer cells and decreased the expression of prostate-specific antigen (PSA), suggesting potential roles as tumor suppressors in this setting. To identify targets of these miRNAs, we combined computational prediction of potential targets with experimental validation by microarray and polyribosomal loading analysis. Three direct targets of the miR-99 family that were validated in this manner were the chromatin-remodeling factors SMARCA5 and SMARCD1 and the growth regulatory kinase mTOR. We determined that PSA is posttranscriptionally regulated by the miR-99 family members, at least partially, by repression of SMARCA5. Together, our findings suggest key functions and targets of miR-99 family members in prostate cancer suppression and prognosis. Cancer Res; 71(4); 1313–24. ©2011 AACR.