SETDB1 has been established as an oncogene in a number of human carcinomas. The present study was to evaluate the expression of SETDB1 in prostate cancer (PCa) tissues and cells and to preliminarily investigate the role of SETDB1 in prostate tumorigenesis in vitro. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to detect the expression of SETDB1 in PCa tissues, adjacent normal tissues, benign prostatic hyperplasia (BPH) tissues, PCa cell lines and normal prostate epithelial cells. The results suggested that SETDB1 was upregulated in human PCa tissues compared with normal tissues at the mRNA and protein levels. The role of SETDB1 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that downregulation of SETDB1 by siRNA inhibited PCa cell growth, and induced G0/G1 cell cycle arrest. The PCa cell migration and invasion decreased by silcencing SETDB1 which were assessed by using in vitro scratch and transwell invasion assay respectively. Our data suggested that SETDB1 is overexpressed in human PCa. Silencing SETDB1 inhibited PCa cell proliferation, migration and invasion.