Runt-related transcription factor 3 (RUNX3) has been reported to be a candidate tumor suppressor gene in gastric cancer. However, in esophageal cancer, the role of RUNX3 has not been studied. The expression of RUNX3 mRNA was quantified by real-time reverse transcription polymerase chain reaction using Taq Man PCR in 15 esophageal cancer cell lines (TE1-15) and 70 esophageal squamous cell carcinoma (ESCC) specimens and their paired normal esophageal mucosa. The data were analyzed with reference to clinicopathological factors. Using specific primers, methylation of the promoter region of RUNX3 was examined. RUNX3 mRNA expression in ESCC tissue was significantly lower than that in the corresponding normal esophageal mucosa (3.913+/-4.617 vs. 7.795+/-15.361, P=0.0345). RUNX3 mRNA expression levels in locally invasive T4 tumors were significantly lower than those in less invasive T1-3 tumors (P=0.0454). Patients who had low RUNX3 mRNA expression levels had a significantly shorter survival after surgery compared with patients who had high RUNX3 mRNA expression (P=0.0299). Among the 15 esophageal cancer cell lines studied, one had methylation of the promoter region of RUNX3. Only 4 in 70 ESCC tumors had methylation in this region. In conclusion, RUNX3 expression may be involved in the tumor invasion and poor prognosis of patients with ESCC. The methylation of the RUNX3 promoter region in esophageal cancer is rare. A study on the mechanisms that underlie the reduced expression of RUNX3 in ESCC is warranted.