In a retrospective analysis of 691 prostate cancer patients, treated with definitive radiotherapy (RT) at the University of Chicago, statin use was significantly associated with improved biochemical control and relapse-free survival. The purpose of this study was to evaluate the effect of simvastatin alone, as well as in combination with radiation, in human prostate cancer cell lines and tumor xenografts. To determine the effect of statin therapy on human prostate cancer cell lines in vitro, clonogenic assays were performed, using escalating simvastatin doses (range, 0 - 20 μM), in PC3 and DU145 cell lines. Clonogenic assays were performed in PC3 cells to study the effect of adding 0.5 μM to varying RT doses (1, 3, 5, and 7 Gy). To study simvastatin as a radiosensitizer in vivo, PC3 and DU145 xenografts in nude mice were treated with: 1) no treatment-control; 2) RT alone (six fractions of 5 - 6 Gy); 3) 200 μg daily simvastatin; or 4) 200 μg daily simvastatin + RT (six fractions of 5 - 6 Gy). Tumor growth delay assays were performed, with five xenografts per treatment group. To investigate the mechanism of simvastatin radiosensitization, DNA expression profiling with Affymetrix Gene-chip of PC3 xenografts was performed for the four treatment arms. A fold-change cut-off of 1.4 was used, with filtration by median values, using significance analysis of microarrays. Clonogenic assays in PC3 and DU145 cell lines demonstrated decreased surviving fractions with escalating simvastatin concentrations. Normalized surviving fractions in PC3 cells were as follows: 0.05 μM, 0.94; 0.2 μM, 0.87; 0.3 μM, 0.55; 0.4 μM, 0.59; 5 μM, 0.0013. Normalized surviving fractions in DU145 cells were as follows: 0.1 μM, 0.42; 0.5 μM, 0.30; 1 μM, 0.21; 5 μM, 0.11; 10 μM, 0.14; 20 μM, 0.099. Clonogenic assays in PC3 cells treated with escalating RT doses ± 0.5 μM simvastatin demonstrated decreased surviving fractions with simvastatin treatment at all RT doses. In PC3 and DU145 xenografts, simvastatin alone had no effect on tumor growth, but simvastatin + RT enhanced tumor response over radiation alone (PC3, p<0.05; DU145, p = 0.056). DNA expression profiling revealed upregulation of multiple Ras/Rho signaling genes with the addition of simvastatin to RT: ARHGAP29, RAP2C, RAB28, RABL2A, RANGNRF, and RGNEF. Simvastatin has antitumor activity in human prostate cancer cell lines, with a clear dose response, and acts as a radiosensitizer in vitro and in vivo. Expressional profiling implicates Ras/Rho signaling to be involved in the radiosensitizing mechanism. The role of Rho proteins in simvastatin radiosensitization is currently under investigation. The use of simvastatin, simvastatin dose escalation, and simvastatin for radiosensitization in prostate cancer treatment should be further studied.