Background: Transurethral resection of bladder tumours (TURBT) is the initial diagnostic treatment for patients with bladder cancer. TURBT is not an easy procedure to master and simulator training may play a role in improving the learning curve. Objective: To implement a national training programme for simulation-based mastery learning in TURBT and explore operating theatre performance after training. Design, setting, and participants: From June 2019 to March 2021, 31 doctors at urology departments in Denmark performed two pretraining TURBT procedures on patients, followed by proficiency-based mastery learning on a virtual reality simulator and then two post-training TURBTs on patients. Outcome measurements and statistical analyses: Operating theatre performances were video-recorded and assessed by two independent, blinded raters using the Objective Structured Assessment for Transurethral Resection of Bladder Tumours Skills (OSATURBS) assessment tool. Paired-sample t tests were used to compare pretraining and post-training analyses and independent t tests for between-group comparisons. This trial is registered at ClinicalTrials.gov as NCT03864302. Results and limitations: Before training, novices had significantly lower performance scores in comparison to those with intermediate experience (p = 0.017) and experienced doctors (p < 0.001). After training, novices significantly improved their clinical performance score (from 11.4 to 17.1; p = 0.049, n = 10). Those with intermediate experience and experienced doctors did not benefit significantly from simulator training (p = 0.9 and p = 0.8, respectively). Conclusions: Novices improved their TURBT performance in the operating theatre after completing a proficiency-based training programme on a virtual reality simulator. Patient summary: We trained surgeons in an operation to remove bladder tumours using a virtual reality simulator. Novice doctors improved their performance significantly after the training, but the training effects for more experienced doctors were minimal. Therefore, we suggest the introduction of mandatory simulator training in the residency programme for urologists.