BACKGROUND AND PURPOSE: A sub-fractionation workflow to correct for intrafraction motion in localized prostate cancer radiotherapy was implemented at our center, allowing for PTV margin reduction from isotropic 5 mm to 2 mm in cranio-caudal and left-right directions and 3 mm in the anterior-posterior direction. The purpose of this study was to assess differences in acute toxicity before and after margin reduction. MATERIALS AND METHODS: Included patients were treated with 36.25 Gy in five fractions on a 1.5 T MR-linac, with PTV margins of 5 mm (standard margins) or 2-3 mm (tight margins). The primary endpoint was acute (90 days post-RT) toxicity. Physician-reported toxicity was measured by maximum CTCAE version 5.0 genitourinary (GU) and gastrointestinal (GI) scores. Patient reported toxicity was a secondary endpoint, assessed through EPIC-26 urinary and bowel domain scores. Groups were balanced through propensity score matching after multiple imputation using chained equations. Pearson's Chi-squared tests were used to analyze CTCAE scores and Wilcoxon rank sum tests to analyze EPIC-26 scores. RESULTS: 299 eligible patients were identified (193 and 106 in the tight and standard margin groups, respectively). After matching, 212 patients (106 per treatment group) were available for assessment. No statistically significant between-group differences in physician-reported toxicity were observed at any follow-up point. Patient-reported urinary irritative/obstructive quality of life was statistically, but not clinically, significantly higher after one month. Overall, scores declined during treatment and one month post-RT, but returned to baseline levels three months post-RT. CONCLUSION: Margin reduction below 5 mm did not seem to reduce acute toxicity after radiotherapy with a stereotactic body radiotherapy (SBRT) treatment schedule in localized prostate cancer. The introduction of real-time comprehensive motion management with prostate gating could further lower GU and GI toxicity and ameliorate treatment related quality of life.