Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors with a poor prognosis. 4-nitroquinoline-1-oxide (4NQO) is a water-soluble quinoline derivative that can successfully induce the production of squamous cell carcinomain vivo. Establishing and optimizing experimental methods for 4NQO induced ESCC formation in mice can provide a more suitablein situmodel for the study of ESCC.