This study sought to determine if runt-related transcription factor 3 (RUNX3) methylation could accurately predict the overall survival in patients with muscle invasive bladder cancer (MIBC). Forty-seven patients with a longer followup period (cohort 1; reported previously) were analyzed. Tumor samples (cohort 2; newly collected) were obtained from 139 MIBC patients. The prognostic significance of RUNX3 methylation was evaluated by Kaplan-Meier analysis and a multivariate Cox regression model. RUNX3 methylation affected the overall survival in cohort 1 (log-rank test; P=0.030). Among the patients in cohort 2, RUNX3 promoter methylation was observed in 93 of the 139 tumor samples (66.9%). Kaplan-Meier estimates showed a significant difference in overall survival according to RUNX3 methylation (P=0.020). By multivariate Cox regression analysis, positive RUNX3 methylation was an independent predictor of overall survival. These results suggest that RUNX3 promoter methylation is a significant prognostic factor for overall survival in MIBC patients.