Background: Radiotherapy impacts the local immune response to cancers. Prostate Stereotactic Body Radiotherapy (SBRT) is a highly focused method to deliver radiotherapy often used to treat prostate cancer. This is the first direct comparison of immune cells within prostate cancers before and after SBRT in patients.Methods: Prostate cancers before and 2 weeks after SBRT are interrogated by multiplex immune fluorescence targeting various T cells and macrophages markers and analyzed by cell and pixel density, as part of a clinical trial of SBRT neoadjuvant to radical prostatectomy.Results: Two weeks after SBRT, CD68, and CD163 macrophages are significantly increased while CD8 T cells are decreased. SBRT markedly alters the immune environment within prostate cancers.