The aim of our current study was to establish an orthotopic inoculation model for studying invasion and metastasis of esophageal squamous cell carcinoma (SCC). Male BALB/c nude mice were used for the experiment. A midline incision was made from the upper to middle abdomen. The abdominal esophagus was carefully exposed. Human esophageal T.Tn SCC cells or human cervical HeLa SCC cells, were injected into the submucosa of the lower esophagus. One of the mice injected with T.Tn cells was sacrificed at 5 weeks, and the remaining five sacrificed at 13 weeks after inoculation. The mice injected with HeLa cells were sacrificed at 3-4 weeks after inoculation. T.Tn cells and HeLa cells formed tumors at the esophagus, but did not metastasize to lymph nodes or lungs. HeLa cells produced peritoneal implants, and directly invaded the stomach and the liver. In the present study, we established a novel orthotopic inoculation model of esophageal SCC. This system is an appropriate and a useful model for studying invasion and metastasis of esophageal SCC, and can also be used as a model for developing therapeutic strategies for esophageal cancer in vivo.