Abstract: Objective To investigate the active status of Notch1 signaling pathway in esophageal squamous cell carcinoma (ESCC) and its effect on cell cycle. Methods The expression of Notch1 gene was detected by immunocytochemistry in EC9706 cells, and the active status of Notch1 signaling pathway was investigated by immunoflurescence. In addition, cell proliferation assay was performed using CCK-8 Kit. Proteins and mRNA of the genes related to cell cycle was studied by Western blot and RT-PCR techniques. Finally, cell cycle distribution was measured by flow cytometry (FCM). Results The expression of Notch1 gene was found in EC9706 cells transfected with pcNICD. Besides, the result of immunoflurecence revealed that Notch1 appeared to be expressed in the cytosol and nucleus, implying an activated status. EC9706 cells had an obvious decreased growth rate compared to the control EC9706 cells and EC9706 cells transfected with pcDNA3.1 (P<0.01). Furthermore, the mRNA and proteins expressions of the CDK2, cyclin D1 and cyclin E genes were significantly decreased in the EC9706 cells transfected with pcNICD compared to those of the cells untreated and transfected with pcDNA3.1. There were higher proportions of cells (74.5%,P<0.05)in EC9706 cells transfected with pcNICD at G₀/G₁ phase than that in cells untreated (59.1%) and transfected with pcDNA3.1 (59.0%). Cell cycle distribution analysis demonstrated that the cell increased (P<0.05)at G0/G1 phase in the cells transiently expressing NICD, which suggested that active Notch1 signaling pathway induces G₀/G₁ cell cycle arrest in EC9706 cells. Conclusion Active Notch1 signaling pathway can obviously inhibit the growth of EC9706 cells, suggesting Notch1 gene may be a new target of treating esophageal squamous cell carcinoma.