Here, we show increased SUB1 expression in prostate cancer cell lines and tissues. Through gene knockdown studies, we demonstrate that SUB1 has an important role in prostate cancer cell proliferation and invasion both in vitro and in vivo. We investigated the role of miR-101 in regulating SUB1 expression. Furthermore, our studies reveal that SUB1 can regulate several oncogenes including therapeutic targets such as PLK1, BUB1B and C-MYC by directly binding to their promoters. Finally, our studies indicate that SUB1-mediated oncogenic phenotype can be reversed by blocking PLK1 activity using PLK1 inhibitor.