We sought to determine if single-dose external beam radiation therapy (EBRT) could modulate the expression signature of T-cell costimulatory and coinhibitory molecules in human prostate cancer (PCa) cell lines in vitro. We investigated the functional impact of irradiated PCa cells with a modulated costimulatory profile on responder T-cell activity. We used three PCa cell lines (DU145, PC3, and LNCaP) and two epithelial cell lines from noncancerous prostate and lung tissue. After 72 hours of EBRT, surface expression of four immunostimulatory molecules (CD70, CD275/ICOSL, CD134L/OX40L, and CD137L/41BBL) and two immunosuppressive markers (CTLA-4/CD152 and PD-L1/CD274) were evaluated by flow cytometry. We evaluated the impact of several radiation doses and the longevity of modulated expression. We examined the functional impact of radiation-induced modulation of cancer cells by cytotoxic T cells (CTL) cytotoxicity and ELISPOT assay for interferon-gamma (IFN-γ) production. Last, we evaluated whether IFN-γ-induced PD-L1 expression could be reversed by EBRT. After 10 Gy EBRT, expression of OX40L and 41BBL increased in all three PCa cell lines; expression of CD70 and ICOSL increased in PC3 cells. Conversely, a decrease in PD-L1 expression in DU145 and PC3 cells was detectable up to 144 hours after EBRT. No PD-L1 was detected in LNCaP. Epithelial cells from normal prostate were not modulated by radiation. CTL cytolytic activity and IFN-γ production were enhanced by interaction with irradiated PCa cells. Finally, EBRT failed to prevent IFN-γ-induced upregulation of PD-L1. We demonstrate that a single dose of EBRT increased surface expression of costimulatory molecules and decreased the expression of coinhibitory molecules in human PCa cell lines. Changes in irradiated tumor cells led to functional enhancement of T-cell activity, despite EBRT failing to reduce IFN-γ-induced expression of PD-L1. These data suggest that combining radiotherapy with T-cell stimulating immunotherapy may be an attractive strategy for cancer treatment.