Runt-related transcriptional factor gene 3 (RUNX3) belongs to the runt domain family of transcriptional factors that plays an important role during normal tissue development and in tumorigenesis in several organs. This study examined the expression of RUNX3 protein in human esophageal mucosa and squamous cell carcinoma in comparison with clinicopathological profiles. Western blot analysis and RT-PCR revealed that both RUNX3/P44 and P27, but not P46, were expressed in all three human esophageal squamous cell carcinoma (SCC) cell lines, as well as in three pairs of esophageal SCC cell lines and the corresponding nontumoral mucosa specimens. RUNX3 expression was shown in prickle and functional cell layer cells in normal esophageal mucosa. On the other hand, immunoreactivity was seen only in carcinoma cells around the cancer pearls. RUNX3 expression was significantly higher in the 19 well-differentiated SCCs than in the 56 moderately or 69 poorly differentiated SCCs (p < 0.01). The 3-year survival rate was significantly lower in the 29 patients with lower RUNX3 expression than in the 37 patients with higher expression (p = 0.0003). These results indicated that RUNX3 protein might play an important role in cellular differentiation in both esophageal mucosa and SCC. The expression correlated with the patients' prolonged survival, implying a tumor suppressive effect in esophageal SCCs.