Background: Muscle-invasive urothelial carcinoma (MIUC) is an aggressive disease with high relapse rates. Neoadjuvant platinum-based chemotherapy (NAC) is the standard of care in patients (pts) who are cisplatin (Cis)-eligible. However, many pts are Cis-ineligible or have persistent muscle-invasive disease after NAC and surgery. We evaluated pembrolizumab (Pembro) as adjuvant therapy in pts with high-risk MIUC following surgical resection. Methods: The AMBASSADOR study is an open-label, randomized, phase III trial that enrolled pts with histologically confirmed MIUC of the bladder, upper tract, or urethra who (1) had ≥ pT2 and/or pN+ or margins+ at surgery (radical cystectomy, nephrectomy, nephroureterectomy, or ureterectomy) following NAC or (2) ≥ pT3 and/or pN+ or margins+ at surgery without NAC and were either Cis-ineligible or declined adjuvant Cis-based therapy. Pts were registered ≥4 to 16 weeks after surgery and randomized 1:1 to receive Pembro 200 mg every 3 weeks for 1 year or observation (Obs). Randomization was stratified by pathologic stage, centrally tested PD-L1 status, and prior NAC. The dual primary endpoints were disease-free survival (DFS) and overall survival (OS) with a target sample size of 739 pts and required 387 DFS and 320 OS events for final analysis. Secondary objectives included evaluation of DFS and OS in PD-L1-positive and -negative pts and assessing safety. Here we present the interim DFS and OS results. Results: Between 09/2017 and 08/2021 a total of 702 pts were randomized prior to early closure due to US FDA approval of nivolumab for MIUC pts: 354 pts to Pembro and 348 pts to Obs; 13.0% vs 21.6% withdrew from study without event in the Pembro vs Obs arms, respectively. 74 pts (21%) in the Obs arm received an immune checkpoint inhibitor. Median follow-up was 22.3 months (mos) for DFS and 36.9 mos for OS. The DFS endpoint (based on 319 events needed for interim analysis) crossed the efficacy boundary. Median DFS was 29.0 months (95% confidence interval (CI) 21.8‒not evaluable (NE)) with Pembro and 14.0 months (95% CI 9.7‒20.20) with Obs (hazard ratio (HR) 0.69 [95% CI 0.55–0.87]; p=0.0013). At the interim analysis (n = 257 events), median OS was 50.9 months (95% CI 43.9‒NE) with Pembro and 55.8 months (95% CI 53.3‒NE) with Obs (HR 0.98 [95% CI 0.76–1.26]; p=0.88). Grade 3+ adverse events occurred in 48.4% and 31.8% of pts in the Pembro and Obs arms, respectively. Conclusions: Adjuvant Pembro demonstrated a statistically significant and clinically meaningful improvement in DFS vs Obs for high-risk MIUC pts after radical surgery. The OS endpoint may have been impacted by pts on the Obs arm receiving a checkpoint inhibitor. Pembro was tolerable with no new safety signals. These results support adjuvant Pembro as a new therapeutic option for pts with MIUC with high risk for recurrence. Additional follow-up is ongoing for the final DFS/OS, PD-L1 subgroups, and ctDNA analyses. Clinical trial information: NCT03244384.