Transforming growth factor-β1 (TGF-β1) acts as a tumor promoter in advanced prostate cancer (PCa). We speculated that microRNAs (miRNAs) that are inhibited by TGF-β1 might exert anti-tumor effects. To assess this, we identified several miRNAs downregulated by TGF-β1 in PCa cell lines and selected miR-3691-3p for detailed analysis as a candidate anti-oncogene miRNA. miR-3691-3p was expressed at significantly lower levels in human PCa tissue compared with paired benign prostatic hyperplasia tissue, and its expression level correlated inversely with aggressive clinical pathological features. Overexpression of miR-3691-3p in PCa cell lines inhibited proliferation, migration, and invasion, and promoted apoptosis. The miR-3691-3p target genes E2F transcription factor 3 (E2F3) and PR domain containing 1, with ZNF domain (PRDM1) were upregulated in miR-3691-3p-overexpressing PCa cells, and silencing of E2F3 or PRDM1 suppressed PCa cell proliferation, migration, and invasion. Treatment of mice bearing PCa xenografts with a miR-3691-3p agomir inhibited tumor growth and promoted tumor cell apoptosis. Consistent with the negative regulation of E2F3 and PRDM1 by miR-3691-3p, both proteins were overexpressed in clinical PCa specimens compared with noncancerous prostate tissue. Our results indicate that TGF-β1-regulated miR-3691-3p acts as an anti-oncogene in PCa by downregulating E2F3 and PRDM1. These results provide novel insights into the mechanisms by which TGF-β1 contributes to the progression of PCa.