MicroRNAs (miRs) are a novel class of RNAs with important roles in regulating gene expression. To identify miRs controlling prostate tumor progression, we utilized unique human prostate sublines derived from the parental P69 cell line, which differ in their tumorigenic properties in vivo. Grown embedded in laminin-rich extracellular matrix (lrECM) gels these genetically-related sublines displayed drastically different morphologies correlating with their behaviour in vivo. The non-tumorigenic P69 subline grew as multicellular acini with a defined lumen and basal/polar expression of relevant marker proteins. M12, a highly tumorigenic, metastatic derivative, grew as a disorganized mass of cells with no polarization, whereas the F6 subline, a weakly tumorigenic, non-metastatic M12 variant, reverted to acini formation akin to the P69 cell line. These sublines also differed in expression of vimentin, which was high in M12, but low in F6 and P69 sublines. Analysis of vimentin’s conserved 3′-UTR suggested several miRs that could regulate vimentin expression. The lack of miR-17-3p expression correlated with an increase in vimentin synthesis and tumorigenicity. Stable expression of miR-17-3p in the M12 subline reduced vimentin levels 85% and reverted growth to organized, polarized acini in lrECM gels. In vitro motility and invasion assays suggested a decrease in tumorigenic behaviour, confirmed by reduced tumor growth in male athymic, nude mice dependent on miR-17-3p expression. Analysis of LCM-purified clinical human prostatectomy specimens confirmed that miR-17-3p levels were reduced in tumor cells. These results suggest that miR-17-3p functions as a tumor suppressor, representing a novel target to block prostate tumor progression.