Aim: To investigate the expression and the clinical significance of runt-related transcription factor (RUNX2) and High mobility group box 1 (HMGB1) in urothelial carcinoma of the urinary bladder. The correlation between RUNX2 and HMGB1 expression and the clinicopathological features was analyzed. Methods: RUNX2 and HMGB1 expression was examined by immunohistochemistry in 62 urothelial carcinoma and 20 non-neoplastic bladder tissues as control group. Results: High RUNX2 and HMGB1 proteins expression was observed in 67.7% and 56.5% of cases urothelial carcinoma respectively and showed predominantly nuclear expression. The expression of RUNX2 and HMGB1 in urothelial carcinoma was significantly higher than that in non-neoplastic urothelial tissues (P<0.001 for each). Moreover, high RUNX2 expression was significantly associated with high tumor grade (P=0.003), advanced tumor stage (P=0.011), lymph node metastasis (P=0.045) and the presence of concomitant carcinoma in situ (CIS) (P=0.01). HMGB1 showed higher expression with high grade tumors (P=0.007), advanced tumor stage (P=0.002) and the presence of necrosis (P<0.001). A significant direct relationship was detected between RUNX2 and HMGB1expression (P< 0.001). Conclusion: Our data suggest that RUNX2 and HMGB1 overexpression may contribute to urothelial carcinoma tumorigenesis and was associated with of aggressive clinicopathologic features. RUNX2 and HMGB1 could be promising potential biomarkers for urothelial carcinoma.