Chemoresistance is a serious problem for the treatment of androgen-independent prostate cancer (PC). The underlying molecular mechanisms by which androgen-independent PC cells acquire the capacity to proliferate remain largely unclear. The aim of this study was to investigate the biological role of prostaglandin reductase 1 (PTGR1) in prostate cancer. Data from the Oncomine database showed that PTGR1 is commonly upregulated in PC tissue in comparison to corresponding normal controls. Two PTGR1-specific short hairpin RNA (shRNA) sequences were used to block the expression of PTGR1 via a lentivirus-mediated system in the androgen-independent PC cell lines DU145 and PC 3. Functional analysis revealed that knockdown of PTGR1 significantly inhibited proliferation and colony formation by PC cells. The inhibition of cell proliferation was related to arrest of the cell cycle in the G0/G1 phase and increased apoptosis in response to PTGR1 knockdown as indicated by flow cytometry. PTGR1 silencing was found to mechanically enhance the expression of p21, caspase 3, and cleaved PARP and to decrease the level of cyclin D1. In conclusion, PTGR1 plays an essential role in PC cells and may be a potential therapeutic target for PC.