Background: Pre-Prostatectomy MRI-Guided Stereotactic Body Radiotherapy for High-Risk Prostate Cancer Trial (PREPARE SBRT; NCT03663218) is an ongoing clinical trial testing the safety of preoperative MRI-guided SBRT for men with clinically localized high-risk prostate cancer. We sought to compare transcriptomic profiles of biopsy (Bx) specimens to acutely irradiated radical prostatectomy (RP) specimens. Methods: Biopsy and post-irradiated RP specimens from 10 subjects were examined. The median interval between the end of RT and the date of RP was 5 days. Transcriptomic profiles were generated using Decipher GRID (Veracyte, Inc). Differences in transcriptional signatures were assessed between Bx samples taken before irradiation and RP samples post-irradiation using a paired T-Test. A control cohort of transcriptomic profiles of 803 untreated Bx samples and matching RP samples from the same patients were used to account for effects in tissue preservation from the procedure different of Bx vs RP. Linear regression model with the interaction effect of cohort (SBRT vs control) by procedure (Bx vs RP) to select signatures impacted by radiation treatment. Results: Transcriptomic signatures associated with Androgen Receptor (AR) activity were significantly downregulated (p=0.002) in irradiated RP specimens compared with pre-treatment biopsies. Analysis of DNA damage repair (DDR) pathways demonstrated that nucleotide excision repair (NER) and non-homologous end joining (NHEJ) signatures were increased (p=0.002 and p=0.02, respectively) after pre-operative RT whereas homologous recombination (p=0.06) and mismatch repair (p=0.08) were not significantly different between pre-treatment and irradiated tissues. Several metabolic-associated pathways were impacted by pre-operative RT including increased glycogen metabolism (p=0.008) and a NADH-NADPH conversion (p=0.001) signature indicative of RT-induced oxidative damage. Consistent upregulation of transcriptomic features associated with increased angiogenesis (p=0.0012) and stromal remodeling (p=0.0008) was observed in irradiated samples. Conclusions: Paired transcriptomic analysis following pre-operative RT demonstrated differential upregulation of specific DDR pathways including NHEJ and NER and metabolic alterations related to oxidative stress and glycogen metabolism. AR activity signatures were decreased in response to pre-operative RT.