Abstract Purpose: To identify genes whose depletion is detrimental to Pim1-overexpressing prostate cancer cells and to validate this finding in vitro and in vivo. Experimental Design: RNAi screening was used to identify genes whose depletion is detrimental to Pim1-overexpressing cells. Our finding was validated using shRNA or PLK1-specific inhibitor BI 2536. Xenograft studies were performed using both PLK1-knockdown cells and BI 2536 to investigate the effects of PLK1 inhibition on tumorigenesis in Pim1-overexpressing cells. Finally, PLK1 and PIM1 expression patterns in human prostate tumors were examined by immunohistochemistry using tissue microarrays. Results: We identified the mitotic regulator polo-like kinase (PLK1) as a gene whose depletion is particularly detrimental to the viability of Pim1-overexpressing prostate cancer. Inhibition of PLK1 by shRNA or BI 2536 in Pim1-overexpressing prostate cancer xenograft models resulted in a dramatic inhibition of tumor progression. Notably, Pim1-overexpressing cells were more prone to mitotic arrest followed by apoptosis due to PLK1 inhibition than control cells. Furthermore, inhibition of PLK1 led to the reduction of MYC protein levels both in vitro and in vivo. Our data also suggest that PIM1 and PLK1 physically interact and PIM1 might phosphorylate PLK1. Finally, PLK1 and PIM1 are frequently co-expressed in human prostate tumors, and co-expression of PLK1 and PIM1 was significantly correlated to higher Gleason grades. Conclusions: Our findings demonstrate that PIM1-overexpressing cancer cells are particularly sensitive to PLK1 inhibition, suggesting that PIM1 might be used as a marker for identifying patients who will benefit from PLK1 inhibitor treatment. Clin Cancer Res; 20(12); 3211–21. ©2014 AACR.