Prostate cancer is the most common solid cancer in men living in the United States. Grapefruit extract contains natural chemicals known as phytochemicals, which include flavonoids and vitamins, which are known to have anti-cancer properties. Previously, our laboratory found that resveratrol, a natural phenol, acts as a radiosensitizer for prostate cancer. Here, we investigate if grapefruit functions similarly. A clonogenic cell survival assay was used to investigate the role of grapefruit extract on survival of a prostate cancer cell line. Following pretreatment with 0-or 50 ng/mL grapefruit extract, the cells were irradiated with 4 grays of radiation. Pretreatment with grapefruit extract resulted in a ~60% reduction in the number of colonies compared to the control. To identify the possible molecular mechanism correlating with the observed decrease in proliferation, we analyzed mRNA expression using a reverse-transcriptase polymerase chain reaction protocol. The anti-proliferative effect of radiation therapy with grapefruit extract pretreatment correlated with the significant downregulation of mRNA expression of the following proteins involved in cell cycle regulation: cyclin B, cyclin D, cyclin-dependent kinase 2, and cyclin-dependent kinase 4. Our data indicate that the combination of grapefruit extract and radiation therapy decreases cell proliferation in PC-3 prostate cancer cells, which suggests a possible role for grapefruit as a radiosensitizer of prostate cancer.