Aberrant activation of the Notch signaling plays an important role in progression of esophageal squamous cell carcinoma (ESCC) and may represent a potential therapeutic target for ESCC. FLI-06 is a novel Notch inhibitor, preventing the early secretion of Notch signaling. However, little information about the antitumor activity of FLI-06 has been reported so far. To evaluate the anti-tumor activity and possible molecular mechanism of FLI-06 to ESCC cells, the effects of FLI-06 on cell viability, apoptosis and cell cycle were evaluated by CCK-8 and flow cytometry assays, respectively, in ESCC cell lines ECa109 and EC9706, and the expressions of proteins in Notch signaling pathway and LSD1 were investigated after cells were treated with FLI-06 by Western blotting. The results showed that FLI-06 blocked proliferation, induced apoptosis and G₁ phase arrest of ESCC cells in a dose-dependent manner. Mechanistically, we found FLI-06 could inhibit Notch signaling pathway by decreasing the expressions of Notch3, DTX1 and Hes1. Interestingly, we also found that the expression of LSD1 (histone lysine specific demethylase 1), which is dysregulated in multiple tumors, was also inhibited by FLI-06. In addition, inhibition of Notch pathway by γ-secretase inhibitor GSI-DAPT could also inhibit LSD1 expression. The current study demonstrated that FLI-06 exerts antitumor activity on ESCC by inhibiting both LSD1 and Notch pathway, which provides the theory support for the treatment of ESCC with FLI-06.