It has been suggested that microRNA-21 (miR-21) functions as an oncogene, as it is overexpressed in many types of tumors compared to adjacent normal tissues. However, the role of miR-21 has yet to be studied in esophageal squamous cell carcinoma (ESCC). miR-21 expression was quantified by real-time reverse transcription polymerase chain reaction in 38 ESCC specimens and their paired non-cancerous mucosa, and in 15 esophageal cancer cell lines (TE1-15). miR-21 expression levels in ESCC tissue were significantly higher than in the corresponding non-cancerous mucosa (6.873±12.664 vs. 1.000, p<0.0001). In patients with more advanced (T3 or T4) tumors, miR-21 expression levels were significantly higher than in those with less advanced (T1 or T2) tumors (P=0.0333). miR-21 expression levels in patients with more invasive infiltrative growth pattern (inf) β tumors were significantly higher than in patients with less invasive infα tumors (P=0.0166). Among the cell lines studied, TE9 had the lowest and TE1 the highest expression of miR-21. Using the miRNA precursor or antisense miRNA inhibitor, we studied how the level of miR-21 influences the proliferation of ESCC cells. Cell proliferation of the anti-miR-21-transfected cell line was significantly lower, while that of the pre-miR-21-transfected cell line was significantly higher than in the control. In ESCC, miR-21 expression may be involved in tumor growth and invasion.